Abstracts from the 14th International Symposium on NeuroVirology October 25–28, 2016, Toronto, Ontario, Canada

Journal of NeuroVirology, Sep 2016

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Abstracts from the 14th International Symposium on NeuroVirology October 25–28, 2016, Toronto, Ontario, Canada

J. Neurovirol. Abstracts from the 14th International Symposium on NeuroVirology October 25-28, 2016, Toronto, Ontario, Canada Rachel Abrams 0 1 3 4 5 6 7 Richa Tyagi 0 1 3 4 5 6 7 Wenxue Li 0 1 3 4 5 6 7 Mario Bianchet 0 1 2 4 5 6 7 Avindra Nath 0 1 3 4 5 6 7 (corresponding author: ) 0 1 4 5 6 7 0 University of Pennsylvania, School of Dental Medicine, Department of Pathology , USA 1 Cagla Akay Espinoza , Ping Lin ( 2 Department of Neurology, Johns Hopkins School of Medicine 3 National Institute of Neurological Disorders and Stroke, National Institutes of Health 4 Joseph Albe , Michael Kujawa, Tiffany Thompson, Amy L. Hartman ( 5 Priyanka Chauhan , Shuxian Hu, Wen Sheng, Sujata Prasad, James Lokensgard ( 6 Cheng-Han (James) Chung , Michael Nonnemacher, Brian Wigdahl, William Dampier ( 7 University of Utah, School of Medice, Department of Pathology , USA P2 Role of ATF6b in HIV-Associated Neurocognitive Disorders - Over the course of human evolution, retroviruses have infected cells of the germ line, allowing the genome of the virus to be passed down from parent to offspring. These human endogenous retroviruses (HERVs) make up about 8 % of the genome; however, in most cases multiple mutations have made them inactive. One of the most recently incorporated (HERV-K); however, has been implicated in the development of amyotrophic lateral sclerosis (ALS). Increased expression of HERV-K viral transcripts was observed in the brains of ALS patients and the expression of HERV-K envelope causes damage to motor neurons in vivo. While there are no treatment options for patients with ALS, in some patients with HIV infection that also display an ALS-like syndrome, antiretroviral drugs can reverse the ALS symptoms. Both HIV and HERV-K utilize an aspartic acid protease to process the viral polyprotein to its active components. To determine if HIV protease inhibitors could inhibit HERV-K protease, HIV protease inhibitors were tested in an in vitro HERVK infection model. The compounds tested had a moderate protective effect; however, were less effective than against HIV infection. To investigate the reason for the level of observed inhibition, similarities and differences between HIV and HERV-K protease were examined. There is no crystal structure of the HERV-K protease available, hence comparative modeling using the sequence alignment with HIV protease was performed. A homology model was generated and refined using the Prime program in the Schrodinger Suites Software package. This model was used to compare the active site of HIV protease with that of HERV-K. It can be seen from the model that while the overall structures of the proteases appear quite similar, changes in the active pocket caused by the few amino acid differences may be sufficient to explain the observed reduction in antiviral activity. The underlying mechanism of cognitive impairment and b r a i n i n j u r y i n p a t i e n t s w i t h H I V- a s s o c i a t e d neurocognitive disorder (HAND) on suppressive antiretroviral therapy are not completely understood. However, synaptic injury, neuronal dysfunction, and damage in these patients are partially driven by immune activation and chronic inflammation in response to soluble factors released by HIV-infected and/or activated macrophages as well as a low level of HIV replication in central nervous system (CNS) reservoirs. A majority of these mediators as well as many of the comorbid conditions were shown to induce a ubiquitous cellular response, unfolded protein response (UPR) in vitro and in vivo. We have previously shown UPR activation in post-mortem tissue from HAND patients in vivo. One of the three master UPR initiator proteins, ATF6b, upon cleavage by site-1 and site-2 proteases (S1P and S2P), translocates to the nucleus for transcriptional induction of ER resident chaperones, apoptotic genes, and secretory pathway regulatory genes. We hypothesized that activation of the ATF6 pathway of the UPR contributed to neuronal damage and death observed in HAND. We found that infection of primary human monocyte-derived macrophages (MDMs) with HIV led to the nuclear translocation of the cleaved, thus active, ATF6b (N-ATF6b), which could be blocked by S1P inhibition. We also observed that blocking nuclear accumulation of N-ATF6b in macrophages led to attenuation of death of primary rat cortical neuroglial cultures exposed to supernatants from HIV-infected MDMs. Finally, we found nuclear N-ATF6b accumulation in neurons exposed to HIV-infected MDM supernatants or excitotoxic stimulus. These findings suggest that altered function of ATF6b in several cell types within the CNS might be contributing to neuronal dysfunction and damage in patients with HAND. P3 Rift Valley Fever virus-induced encephalitis: characterization of leukocytes in the CNS Department of Infectious Disease and Microbiology, Center for Vaccine Research, University of Pittsburgh Background: Rift Valley Fever Virus (RVFV) is a vectorborne infection endemic to the Horne of Africa; However recent outbreaks in the Arabian Peninsula have expanded its potential range. People can develop encephalitis as a result of RVFV infection. Our lab uses an immunocompetent Lewis rat model to study neuropathogenesis of RVFV. Lewis rats develop uniformly lethal encephalitis within 7–8 days after aerosol exposure. Rats infected subcutaneously do not develop disease. The goal of this study is to characterize the phenotypes, timing, and extent of immune cell infiltrate into the CNS of RVFV-infected Lewis rats. Methods: Lewis rats were infected with RVFV ZH501 by aerosol and subcutaneous routes, then serially sacrificed between days 3 and 6. Rat immune cells were isolated from brains via percoll gradients. Cell populations were stained with antibodies, run on a BD LSRII flow cytometer, and analyzed with FlowJo 7.6.5. Results: In rats infected by aerosol, infiltration of inflammatory cells into the CNS began at 4 dpi and consisted of neutrophils, macrophages, and lymphocytes. There was also evidence of microglia activation. Rats infected subcutaneously do not develop overt disease yet still had a detectable infiltrate that was intermediate compared to uninfected rats. Neutrophils were a major component in aerosol but not subcutaneously infected rats. Conclusions: In addition to extensive viral replication in the brain, leukocyte infiltration and microglia activation are a significant component of disease in the Lewis rat model of RVFV. The relative contributions of viral cytopathology versus immunopathology remain to be determined, but this study represent the first attempt to characterize the leukocytic component. P4 Selection of gRNAs to target the HIV-1 quasispecies with CRISPR/cas9 Alexander Allen1, Michael Nonnemacher1, William Dampier1,2, Matthew Desimone2, Vanessa Pirrone1, Katie Kercher1, Shendra Passic1, Jean Williams1, Brian Wigdahl1 (corresponding author: ) 1Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine; 2School of Biomedical Engineering and Health Systems, Drexel University HIV-1 viral persistence during long-term antiretroviral therapy is a major hurdle to a cure. Genomic editing techniques, like the CRISPR/Cas9 system, hold promise to permanently excise integrated virus from a host cell. Targets are defined by a 20 nucleotide guide RNA (gRNA) complementary to the desired genomic region. However, due to the rapid mutation rate intrinsic to HIV-1 replication, the virus in patients exists as a collection of distinct genomic variants, termed quasispecies. Presented here is a methodology for designing gRNA sequences to cleave a spectrum of HIV-1 quasispecies present within individual patients across a population of HIV1-infected patients. PBMC genomic DNA was isolated from patients in the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort as well as from brain and spleen tissue from the National NeuroAIDS Tissue Consortium (NNTC) and the long terminal repeat (LTR) of the HIV-1 quasispecies was sampled using Next Generation Sequencing (NGS). gRNAs were computationally selected by examining their binding potential across a random training set of CARES patient samples. A package of 4 or 10 gRNAs were selected based on the training set which in silico cleaved the entire detectable quasispecies of the remaining CARES samples and of the majority of the NNTC samples with an average number of cleavages within each sample group of 3.4+/−1.7 or 5.4+/−3 times, respectively. The package was further tested in silico against a national sampling of subtype B North American LTRs from the Los Alamos National Database and was shown to cleave all sequences. Currently, we are focused on cloning these packages of gRNAs to initiate functional studies to confirm the in silico studies performed to date. These studies represent a step towards understanding the complex task of using excision therapy to target HIV-1 quasispecies in the infected patient population. P5 Functional consequences of HTLV-1 viral antigens detected in exosomes from HAM/TSP patients Monique Anderson1, Yoshimi Enose-Akahata1, Maria Chiara Monaco-Kushner1, Ashley Vellucci1, Yuetsu Tanaka2, Ben Lepene3, Fatah Kashanchi4, Steven Jacobson5 (corresponding author: ) 1National Institutes of Health; 2University of the Ryukyus Graduate School of Medicine; 3Ceres Nanosciences; 4George Mason University Laboratory of Molecular Virology; 5National Institutes of Health Human T-cell lymphotropic virus Type I (HTLV-1) is a retrovirus that currently infects an estimated 10–20 million people worldwide. The vast majority of those infected will remain asymptomatic, but about 0.1–4 % of patients will develop HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP is a neuroinflammatory disease that has been shown to be immunopathologically mediated. Recent data suggests that viral antigens can be acquired in the absence of viral infection through the transport of exosomes. Therefore, the presence of HTLV-1 products in the exosomes isolated from HAM/TSP patient cerebrospinal fluid (CSF) was investigated. Using a novel nanotrap technology which utilizes hydrogel particles that trap exosomes, exosomes that were positive for HTLV-1 Tax by Western blot could be detected from HAM/TSP patient CSF but not from control HTLV-1 seronegative multiple sclerosis (MS) patients. Additionally, HAM/TSP PBMCs were shown to secrete exosomes containing HTLV-1 Tax in ex vivo cultures. To understand the source of these exosomes, PBMCs were sorted and found to have increased exosome production by activated CD4 + CD25+ and CD8 + CD25+ T cells. Importantly, to determine if these exosomes are functionally producing tax proteins, exosomes isolated from HAM/TSP PBMCs were applied to recipient target B cells. These exosome-sensitized target cells were shown to be lysed by HTLV-1 Tax-specific cytotoxic T lymphocytes (CTL). Additionally, initial results indicate that Tax can be introduced into exosomes with transfection of Tax plasmids into uninfected cells, and this process was unaltered with Tax mutation. Cumulatively, these findings show that there are HTLV-1 proteins present in exosomes found in virus-free CSF of HAM/TSP patients and that HAM/ TSP PBMCs can excrete these exosomes in culture. Furthermore, exosomes containing HTLV-1 Tax can sensitize target cells for HTLV-1 specific lysis by CTLs. This suggests that exosomes may play a role in perpetuating the activated immune response that contributes to HAM/TSP. P6 Extracellular vesicles are amyloid carriers in HIV-infected brains Ibolya E. Andríçs1, Ana Leda1, Marta Garcia-Contreras 2, Luc Bertrand1, Minseon Park1, Marta Skowronska1, Michal Toborek1 (corresponding author: ) 1Department of Biochemistry and Molecular Biology, University of Miami School of Medicine; 2Diabetes Research Institute, University of Miami School of Medicine HIV-infected brains are characterized by increased amyloid beta (Abeta) deposition. It is believed that the blood–brain barrier (BBB) is critical for Abeta homeostasis and contributes to Abeta accumulation in the brain. Extracellular vesicles (ECV) gained recently a lot of attention as potentially playing a significant role in Abeta pathology. In addition, HIV-1 hijacks the exosomal pathway for budding and release. Therefore, we investigated the involvement of BBBderived ECV in the HIV-1-induced Abeta pathology in the brain. Our results indicate that HIV-1 increases ECV release from brain endothelial cells and elevate their Abeta cargo as compared to controls. Interestingly, brain endothelial cellderived ECV transferred Abeta to astrocytes and pericytes. Infusion of brain endothelial ECV carrying fluorescent Abeta into the internal carotid artery of mice resulted in Abeta fluorescence associated with brain microvessels and also in the brain parenchyma. These data suggest that ECV carrying Abeta can be successfully transferred across the BBB into the brain. Based on these observations, we conclude that HIV-1 facilitates shedding of brain endothelial ECV carrying Abeta, a process that may increase Abeta exposure of cells of neurovascular unit, such as astrocytes and pericytes. This mechanism may contribute to increased amyloid deposition in HIV-infected brain. Supported by M H 0 9 8 8 9 1 , M H 0 7 2 5 6 7 , D A 0 3 9 5 7 6 , D A 0 2 7 5 6 9 , HL126559, and by the Miami CFAR MH063022. P7 HIV-1 Vpr sequence variants associate specifically with co-linear genetic determinants of viral co-receptor phenotype G r e g o r y A n t e l l 1 , W i l l i a m D a m p i e r 2 , B e n j a m a s Aiamkitsumrit2, Michael Nonnemacher2, Vanessa Pirrone2, Wen Zhong2, Katherine Kercher2, Shendra Passic2, Jean Williams2, Yucheng Liu2, Tony James2, Jeffrey Jacobson3, Zsofia Szep4, Brian Wigdahl2, Fred Krebs2 (corresponding author: ) 1School of Biomedical Engineering, Science, and Health Systems, Drexel University; 2Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine; 3Department of Medicine, Section of Infectious Disease, Lewis Katz School of Medicine, Temple University; 4Division of Infectious Diseases and HIV Medicine, Department of Medicine, Drexel University College of Medicine Viral protein R (Vpr) is a 14 kDa HIV-1 accessory protein that functions in HIV-1 replication and pathogenesis. Vpr is also a likely contributor to the neuropathogenesis that underlies deficits collectively referred to as HIVassociated neurocognitive disorders (HAND). HIV-1 infection of the brain is predominated by infected macrophages and microglial cells that harbor viruses characterized by an R5 (CCR5-utilizing) co-receptor genotype. In studies that have paralleled our identification of sequence signatures in HIV-1 Tat and the long terminal repeat (LTR) that associate with co-receptor utilization, we have demonstrated associations between Vpr variation in HIV1-infected patients and co-receptor usage. Co-linear HIV1 Vpr and Env-V3 amino acid sequences were collected from the LANL HIV-1 sequence database, as well as from well-suppressed patients enrolled in the Drexel/Temple Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. Vpr sequences were grouped as X4 (CXCR4-utilizing) or R5 according to genotypic classification of the co-linear Env-V3 sequence. The amino acid diversity of each Vpr population was assessed, as was the Jensen-Shannon divergence between groups, in order to identify sequence signatures associated with co-receptor utilization. Multiple amino acid alphabets were utilized in order to better assess the impact of amino acid substitutions with similar physiochemical properties. Positions 35–45 and the C-terminus of Vpr consistently displayed statistically significant divergence across multiple alphabets. Specifically, positions 37 and 41 displayed a consensus amino acid switch between R5 and X4 sequence populations. Overall, the results suggest a functional link between the evolution of Vpr and gp120 in HIV-1infected patients, demonstrate the existence of HIV-1 tropism beyond the envelope, and suggest associations between Vpr variants and R5 viral genotypes in the brain. P8 Divergent efficacies of antiretroviral therapy in blood and brain: HIV-1 suppression is dependent on drug and cell type. 1Department of Medicine, University of Alberta; 2Department of Psychiatry, University of Alberta; 3Department of Pathology, University of Calgary; 4Department of Medicine and Psychiatry, University of Alberta Background: Perivascular macrophages and microglia are the principal productive reservoir for active HIV-1 replication in the brain. In patients receiving suppressive combination antiretroviral therapy (cART), HIV-1 replication in the brain reservoir is assumed to be latent or minimally productive although the susceptibility of this reservoir to suppression by cART is uncertain. We investigated the efficacies and concentrations of established antiretroviral drugs (ARVs) in HIV-infected microglia and macrophages together with evaluating the in vivo brain HIV-1 reservoir during suppressive cART. Methods: HIV-1 RNA and DNA levels in brain tissues from HIV-infected patients (n = 2) were quantified by digital droplet PCR within 12 h of last ARV dosage. The efficacies of individual ARVs in HIV-infected primary microglia, bone marrowderived macrophages and peripheral blood mononuclear cells (PBMCs) were compared. Tissue and cellular conc e n t r a t i o n s o f A RV s w e r e m e a s u r e d b y l i q u i d chromatography-mass spectrometry (LC-MS) in human myeloid cells and mouse brain. Results: In treated patients with prolonged (>4 years) undetectable plasma viral RNA levels, HIV-1 RNA and DNA were detected in multiple brain specimens with associated neuroinflammatory responses. Zidovudine, etravirine and darunavir treatment of HIV-infected microglia exhibited significantly higher E C 5 0 v a l u e s t h a n t h o s e o b s e r v e d i n P B M C s . Intracellular and extracellular ARV levels in human myeloid cells were similar and unaffected by HIV-1 infection. In vivo concentrations of darunavir and raltegravir in mice varied depending on brain region and were >1.0 log lower than matched plasma levels at 1 h and undetectable at 8 h post-treatment. Conclusions: ARVs displayed differential concentrations and antiviral effects depending on the individual ARV and infected cell type (or tissue), which was substantiated by the presence of persistent viral DNA and RNA in brain tissue from patients with undetectable plasma viral RNA. These findings underscore considerations of assessing ARV selection for different viral reservoirs as part of efforts to eradicate HIV-1. the HIV genome into overlapping fragments of 1000 (+/−200) base pairs allowing PCR amplification of each fragment. Next generation sequencing is then used to acquire sequence data with sufficient depth to identify genetic variants making up less than 1 % of the quasispecies in the sampled genome, allowing for design of potential CRISPR-cas9-based cure strategies. P9 Identification of HIV-1 quasispecies for targeted excision of latent infection using next generation sequencing 1Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine; 2Division of Infectious Disease, Department of Medicine, Drexel University College of Medicine; 3Department of Medicine, Lewis Katz School of Medicine, Temple University Although ART drastically improves the prognosis for HIV-1infected patients, latent infection due to integrated proviral DNA persists in some tissues (e.g. peripheral blood, lymphoid tissue, and the brain). There is currently no effective strategy for the removal of persistent proviral DNA from infected patients and the end result of ART interruption is a resurgence of systemic viral load. However, recent success has been achieved in the removal of HIV-1 from infected cell lines as well as primary cells from infected patients and the results of these experiments suggest a new approach to the targeted removal of latent infection. Bioinformatic analysis of deep sequencing data can potentially be used to design gRNAs which target persistent HIV DNA using the CRISPR-Cas9 system. In order to pursue this strategy, it is necessary to collect sequence data from patients with ART regulated viral loads. Furthermore, it is critically important to track genetic variation over time in HIV-1-infected patients as the genetic sequence of latent HIV infection is not static. To address this complex problem, we have utilized patient samples from the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. The CARES Cohort collects longitudinal samples from patients allowing observation of the progress of HIV-1 mutation. Presented here are the strategies that we have developed to address the challenge of whole-genome sequencing of latent HIV infection in ART-regulated patients. We have determined that an effective strategy is to subdivide P10 Human neural progenitor cells (hNPCs) are productively infected by R5-tropic HIV-1: Morphine interactions on infection and function involve Cdk5 signaling Joyce Balinang1, Kurt Hauser2, Pamela Knapp1 (corresponding author: ) 1Department of Anatomy and Neurobiology, Virginia Commonwealth University; 2Department of Pharmacology and Toxicology, Virginia Commonwealth University Opiates augment HIV-induced neuropathogenesis in the CNS through both direct and indirect mechanisms that disrupt glial and neuronal function. All CNS macroglia and neurons derive from neural progenitor cells (NPCs) during development, and NPCs in the adult brain contribute to repair processes. Since disruptions in NPC function are known to impact CNS populations and brain function in a number of disease/injury conditions, we examined whether HIV ± opiate exposure affected the maturation and fate of human NPCs. As hNPC infection by HIV has occasionally been reported, we also reexamined this question, and parsed between effects due directly to hNPC infection by HIV, or to hNPC dysfunction caused by the infective milieu. Multiple approaches confirmed the infection of hNPCs by R5 tropic HIVBaL, and demonstrated that active infection could be sequentially transferred to naíve hNPCs. Exposure to supernatant from HIVBaL-infected cells (HIVsup) reduced hNPC proliferation and led to premature differentiation into astrocytes and neurons. Morphine coexposure prolonged hNPC infection and exacerbated functional effects of HIVsup. Neither purified virions nor UVinactivated HIVsup altered proliferation, indicating that this effect did not require infection. Gene array analysis and RTqPCR with immunoblot validation suggested that Cdk5 signaling was involved in HIV-morphine interactions. siRNAmediated knockdown of Cdk5 expression reversed premature MAP2 differentiation, but also increased hNPC death. Pharmacological inhibition of Cdk5 activity with roscovitine also reversed MAP2 differentiation without inducing hNPC death. However, roscovitine also inhibits Cdk2/Cdk4 activity, which may e xplain its antiproliferative effects on hNPCs independent of treatments. Overall, dysregulation of hNPC functions by the infectious environment may create cell population imbalances that contribute to CNS deficits in both adult and pediatric patients. Additionally, infected hNPCs may pass virus to their progeny, and serve as an unappreciated viral reservoir. The recent epidemic of opiate/heroin abuse highlights the clinical importance of HIV and opiate interactions. P11 cAMP signaling enhances HIV-1 long terminal repeat (LTR)-directed transcription and viral replication in human bone marrow progenitor cells Anupam Banerjee, Luna Li, Vanessa Pirrone, Fred Krebs, Brian Wigdahl, Michael Nonnemacher (corresponding author: ) Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine CD34+ hematopoietic progenitor cells have been shown to be susceptible to HIV-1 infection, possibly due to a low level expression of CXCR4, a coreceptor for HIV-1 entry. Given these observations, we have explored the impact of forskolin on cell surface expression of CXCR4 in a cell line model (TF-1). The elevation of intracellular cAMP by forskolin through adenyl cyclase resulted in transcriptional upregulation of CXCR4 with a concomitant increase in replication of the CXCR4-utilizing HIV-1 strain IIIB. Transient expression analyses also demonstrated an increase in CXCR4-, CCR5, and CXCR4-/CCR5-utilizing HIV-1 (LAI, YU2, and 89.6, respectively) promoter activity. Studies also implicated the protein kinase A pathway and the downstream transcription factor CREB-1 in interfacing with cAMP response elements located in the CXCR4 and viral promoter. These observations suggest the cAMP signaling pathway may serve as a regulator of CXCR4 levels and concomitantly of HIV-1 replication in bone marrow progenitor cells. P12 Exosomes from donor and recipient cells: Role of exosomes in HIV latency in cART treated cells. 1National Center for Biodefense and Infectious Disease George Mason University; 2Ceres Nanosciences Inc., Manassas, VA; 3Florida International University, Miami, FL HIV-1 infection results in a chronic illness since long-term HAART can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under HAART frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naíve cells to HIV-1 infection. Up to a million copies of TAR RNA per microliter were also detected in the serum from HIV-1 infected humanized mice suggesting that TAR RNA may be stable in vivo. We recently have found another viral non-coding RNA that we termed TARgag which does not code for a protein but is present in the exosomes. Incubation of exosomes from HIV-1 infected cells with primary macrophages resulted in a dramatic increase of pro-inflammatory cytokines, IL-6 and TNF-beta, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. Furthermore, the single stranded 5’ or 3’ processed stem RNA binding to TLRs activates the NF-kappaB pathway and regulates cytokine expression. In our most recent data, we find that the number of exosomes from infected cells is increased when cells are treated with cART. This directly indicates that HIV viral release and exosome release have overlapping biogenesis pathways, including the ESCRT pathway. We will also discuss exosomes from other neuro-tropic RNA viral infections, including HTLV-1, Ebola, RVFV, and Zika, and how they behave differently than exosomes from HIV infected cells. Collectively, our results imply that exosomes in viral infection control pathogenesis and targeting these particles may be a method to lower overall viral burden in infected hosts. P13 Role of DNA damage response pathways in the activation of polyomavirus JC Anna Bellizzi1,2, Martyn K. White2, Gabriele Ibba2, Kamel Khalili2, Hassen S. Wollebo2 (corresponding author: ) 1Department of Public Health and Infectious Diseases, Institute Pasteur, Cenci-Bolognetti Foundation, Sapienza University, Rome Italy; 2Center for Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, Philadelphia PA Infection of glial cells by the human neurotropic polyomavirus JC (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML) rapidly inflicts damage to cellular DNA, which in turn evokes the DNA damage response (DDR). We previously reported that JCV rapidly induces expression of the DNA repair factor Rad51 and Rad51 activates the JCV early promoter in co-operation with the transcription factor NF-kappaB p65. Thus Rad51 induction by JCV infection serves as a positive feedback mechanism for viral activation early in JCV infection. It is known that the DDR may also stimulate NF-kappaB activity, a phenomenon known as nucleus to cytoplasm or “inside-out” NF-kappaB signaling. This pathway is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase that is recruited and activated by DNA double-strand breaks and subsequently involves a series of post-translational modifications of NF-B essential modulator (NEMO), also known as inhibitor of NF-kappaB kinase subunit gamma (IKK-gamma), which activates NF-kappaB. Here we show that JCV infection causes phosphorylation and activation of ATM while a specific small molecule inhibitor of ATM, KU-55933, inhibits JCV replication. Analysis of the effect of JCV infection on the subcellular distribution of NEMO by cell fractionation and immunocytochemistry showed a redistribution of NEMO from the cytoplasm to the nucleus. Co-expression of JCV large T-antigen and FLAG-tagged NEMO in cells showed the occurrence of the sumoylation of NEMO in the presence of T-antigen, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO. Taken together, we propose a model where JCV infection induces both the expression level of Rad51 protein and nucleus to cytoplasm NF-kappaB signaling, which then act together to enhance viral gene expression. P14 CRISPR/Cas9 system as an agent for eradication of polyomavirus JC infection 1Department of Public Health and Infectious Diseases, Institute Pasteur, Cenci-Bolognetti Foundation, Sapienza University, Rome Italy; 2Center for Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, Philadelphia, PA Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the CNS caused by lytic infection of oligodendrocytes, which produce myelin in the brain, with human neurotropic polyomavirus JC (JCV). PML lesions are areas of demyelination containing oligodendrocytes with viral nuclear inclusion bodies and bizarre astrocytes, which are also productively infected by JCV. Although JCV was isolated over 40 years ago and has been extensively studied since, there is still no effective therapy for PML. Recently, a novel genome-editing method was developed based on clustered regularly interspaced short palindromic repeat (CRISPR) systems. The CRISPR system uses a nuclease, CRISPRassociated (Cas9), that complexes with small RNAs as guides (gRNAs) to cleave DNA in a sequence-specific manner upstream of the protospacer adaptor motif ( PA M ) i n a n y g e n o m i c l o c a t i o n . H e r e , w e u s e CRISPER/Cas9 system as a potential tool for JCV elimination by designing guide RNAs (gRNAs) targeting regions within the early gene encoding viral protein, T-antigen. Our results indicated that Cas9/gRNAs targeted to the JCV T-antigen gene effectively delete target DNA sequence, reduce T-antigen expression and late promoter activity and inhibit JCV DNA replication. No off-target effects of the JCV-specific CRISPR/Cas9 editing were detected using the SURVEYOR assay. These data reveal the promise of CRISPR/Cas9 as a tool for the elimination of the JCV genome and a potential as a cure for PML. P15 Inhibition of HSV-1 replication by a gene editing strategy Anna Bellizzi1,3, Pamela C. Roehm2,3, Masoud Shekarabi3, Hassen S. Wollebo3, Gabriele Ibba3, Martyn K. White3, Lifan He3, Julian Salkind3, Kamel Khalili3 (corresponding author: ) 1Department of Public Health and Infectious Diseases, Institute Pasteur, Cenci-Bolognetti Foundation, Sapienza University, Rome Italy; 2Department of Otolaryngology/ Head and Neck Surgery - Department of Neurosurgery, Lewis Katz School of Medicine at Temple University, Philadelphia PA; 3Center for Neurovirology, Department of Neuroscience, Lewis Katz School of Medicine at Temple University, Philadelphia PA HSV-1 induced illness affects more than 85 % of adults worldwide and there is no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target DNA sequences of the HSV-1 genome for deletion that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 Nterminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases. P16 Mechanisms associated with differential inflammatory effects of Tat proteins derived from HIV-1 subtypes-B and recombinant CRF02_AG on human brain microvascular endothelial cells BIJU BHARGAVAN1, GEORGETTE KANMOGNE1 (corresponding author: ) 1 D e p a r t m e n t o f P h a r m a c o l o g y a n d E x p e r i m e n t a l Neuroscience, University of Nebraska Medical Center Previous studies showed that HIV-1 Tat induce blood–brain barrier (BBB) dysfunction and is involved in HIV-associated neurocognitive disorders (HAND). These studies were performed mostly with Tat from HIV-1 subtype-B (Tat.B), whereas 70 % of the 37 millions HIV-infected subjects are in Sub-Saharan Africa and are infected with non-B subtypes, including the recombinant HIV-1 CRF02_AG, prevalent in West-Central Africa. The effects of this recombinant virus on the BBB and HAND are not known. In the current study, we analyzed the effects of Tat.B and Tat from HIV-1 CRF02_AG (Tat.AG) on primary human brain microvascular endothelial cells (HBMEC), the major component of the BBB. Exposure of HBMEC to Tat.B increased the expression and secretion of the proinflammatory cytokine IL6 by 9-fold (p < 0.001) and increased IL6 mRNA by 113-fold (p < 0.001), whereas Tat.AG increased IL6 expression by 2.7 to 3.8-fold, and increased IL6 mRNA by 35.7-fold (p < 0.001) compared to controls. Mechanistic studies showed that Tat.B induced BBB inflammation through the IRAK1/4,MKK/JNK pathways, in an AP1- and NFkB-independent manner, whereas Tat.AG effects occurred via the MKK/JNK/AP1/NFkB pathways. We further showed that Tat-induced effects were associated with activation of c-jun(serine-63) and SAPK/JNK(Thr183/Tyr185), demonstrated increased expression of transcription factors associated with these pathways (Jun, STAT1, RELB, CEBPA) with higher levels in Tat.B-treated cells compared Tat.AG-treated cells. Functional studies showed that both Tat.B and Tat.AG decreased the expression of endothelial tight junction proteins claudin-5 and ZO-1, and decreased brain trans-endothelial electric resistance (TEER), but greater reduction in TEER was observed with Tat.B compared to Tat.AG. Overall, our data showed increased BBB inflammation and BBB disruption with Tat.B, compared to Tat.G. This may suggests these two HIV-1 subtypes differentially affect the BBB and the CNS. Our future studies will validate these findings by analyzing the direct effects of these viral strains on the BBB and the CNS. P17 Dysregulation of epigenetic control of memory in HIV induced mild cognitive disease in mice and disease relief by valproic acid treatment 1Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, USA HIV infected individuals on suppressive antiretroviral therapy develop chronic mild neurocognitive impairments (mild NCI) that may affect everyday activities but do not progress to HIV dementia. The pathobiology of mild NCI is unclear and there is no treatment, but one feature indicative of disease mechanism is an apparent discordance between broad neuronal dysfunction and limited neuronal death. We investigated NCI biology and treatment in experimentally induced disease in immunocompetent mice infected with chimeric HIV, EcoHIV. We first show in behavioral tests that infected animals develop chronic NCI involving at least two cognitive domains, memory and executive function. The resemblance of murine disease to human mild NCI was indicated by low HIV burdens, mild neuroinflammation, and diffuse dendritic pruning in multiple brain regions but absence of overt neuropathology and cellular apoptosis. We then applied systems biology approach to gain an insight into disease pathobiology. Comparative analysis of brain gene expression profiles from cognitively impaired mice and patients with HIV cognitive disease revealed common downregulation of pathways controlling neuronal signal transmission and memory. Assessment of epigenetic control of these functions by chromatin immunoprecipitation and sequencing indicated high correlation between histone 3 hypermethylation at lysine 9 m3 and transcriptional suppression of genes associated with synaptodendritic functions. Treatment of cognitively impaired mice with valproic acid significantly mitigated murine NCI, normalized selected synaptic gene expression, and reversed histone hypermethylation on their promoters. The results suggest dysregulation of epigenetic control of memory in HIV induced mild cognitive dysfunction and indicate that the disease is treatable. P18 Plasma soluble CD163 is associated with postmortem brain pathology in human immunodeficiency virus infection. 1HIV Neurobehavioral Research Program, San Diego, CA; 2Department of Neuroscience, Temple University, Lewis Katz School of Medicine, Philadelphia, PA; 3Department of Biology, Boston College, Chestnut Hill, MA; 4Department of Psychiatry, University of California San Diego, La Jolla, CA; 5Department of Neurosciences, University of California San Diego, La Jolla, CA; 6Department of Neurology, University of California Los Angeles, Los Angeles, CA Background: Plasma soluble CD163 (sCD163), shed by m o n o c y t e s a n d m a c r o p h a g e s , c o r r e l a t e s w i t h neurocognitive impairment in human immunodeficiency virus (HIV) infection and is a potential biomarker for neurologic damage in HIV. We sought to determine the association between plasma or cerebrospinal fluid sCD163 and postmortem brain pathology. Methods: We measured sCD163 levels in antemortem plasma (n = 54) and cerebrospinal fluid (n = 32) samples from 74 HIV+ participants (median 5 months before death) who donated their brains to research at autopsy. We quantified markers of synaptodendritic damage (microtubule-associated protein 2 [MAP2], synaptophysin [SYP]), microgliosis (HLADR, ionized calcium binding adaptor molecule 1), astrocytosis (glial fibrillary acidic protein) and impaired protein clearance (beta-amyloid) in frontal cortex, hippocampus, putamen, and internal capsule. Multivariable least-squares regression was used to evaluate the association between plasma or cerebrospinal fluid sCD163 and histological measures, accounting for medical comorbidities and correcting for multiple comparisons. Results: Higher plasma sCD163 was associated with lower MAP2 in frontal cortex (B = −0.23, 95 % CI −0.41 to −0.06, p = 0.007), putamen (B = 0.32, 95 % CI −0.52 to −0.12, p = 0.004), and hippocampus (B = −0.23, 95 % CI −0.35 to −0.10, p = 0.007), and with lower SYP in hippocampus (B = −0.25, 95 % CI −0.42 to −0.03, p = 0.003) but not putamen or frontal cortex (p > 0.05). Higher plasma sCD163 was associated with higher HLA-DR in putamen (B = 0.17, 95 % CI 0.08 to 0.26, p < 0.001) and when averaged across three brain regions (B = 0.11, 95 % CI 0.05 to 0.17, p < 0.001). Cerebrospinal fluid sCD163 was not associated with any histological measure (p > 0.05). Conclusion: Higher plasma sCD163 in life is associated with greater synaptodendritic damage (SYP, MAP2) and microglial activation (HLA-DR) in cortical and subcortical brain regions. Plasma sCD163 warrants additional study as a biomarker of neurologic damage in HIV infection. P19 Extracellular vesicle mediated shuttling of miR-9 mediates synaptodendritic alterations in HAND Shilpa Buch, Lu Yang, YeonHee Kook, Yu Cai, Guoku Hu (corresponding author: ) Reversible synaptodendritic injury and underlying neuroinflammation are important phenotypes and correlates of HIVassociated neurocognitive disorders (HAND). Similar to HIV+ subjects on antiretroviral therapy (ART), SIV-infected rhesus macaques on ART have also been shown to exhibit loss of synaptophysin, increased glial activation and dysregulation of various signature microRNAs (miRs). MiR-mediated regulation of disease pathogenesis represents an evolving area of research that has ramifications for identification of potential therapeutic targets for various neurodegenerative disorders, for which, currently there exists no cure. Parallel to the advances made in miRNA research has been the advent of the field of extracellular vesicles (EVs). EVs represent an important mode of intercellular communication by serving as conduits for transfer of membrane and cytosolic proteins and RNA (including miRs) between cells. Despite effective suppression of virus replication in the presence of ART, viral proteins such as HIV Tat have been found to be present in tissues such as the CNS and the lymph nodes, likely contributing to ongoing neuroinflammation. In the current study we demonstrate that exposure of astrocytes to HIV Tat resulted in increased induction/release of miR-9 in the EVs. MiR-9enriched EVs, were in turn, taken up by the neurons resulting in significant synaptodendritic injury. Furthermore, we also observed downregulated expression of miR-9 targets PDGF-CC, NLGN2 and MCPIP1 (key players in regulating the normal synaptic functioning in neurons) in SIV+/HIV+ brains compared with the uninfected controls. Validation of these findings in rat primary neurons infected with lentivirus expressing miR-9 demonstrated decreased expression of the respective targets. Moreover, we also observed reduced expression of these targets and concomitant synaprodendritic injury in the neurons exposed to EVs isolated from conditioned media collected from HIV Tat-treated astrocytes. Taken together, our findings implicate that HIV-Tat mediated alteration of synaptodendritic injury is regulated by exosomal miR-9 via its targets (PDGF-CC/MCPIP1/NLGN2). P20 Neurotoxic lysosomal protease cathepsin B is present in macrophage-derived exosomes and internalized by neurons Yisel Cantres-Rosario1, Karla Negron Roure2, Marines Plaud1, Loyda Melendez3 (corresponding author: ) 1Department of Microbiology and Medical Zoology, School of Medicine, University of Puerto Rico-Medical Sciences Campus; 2Department of Biology, University of Puerto Rico - Bayamon Campus; 3Department of Microbiology and Medical Zoology, School of Medicine & Translational Proteomics Center, University of Puerto Rico-Medical Sciences Campus Despite the efficacy of antiretroviral therapy, mild forms of HIV-associated neurocognitive disorders (HAND) remain prevalent. HIV-infected macrophages infiltrate into the brain secreting viral and cellular proteins that trigger neuronal dysfunction and death. One of the monocyte-derived macrophages (MDM) secreted proteins is cathepsin B, a lysosomal protease that triggers neuronal apoptosis in vitro, and co-localizes with beta-amyloid peptides in brain tissue from HAND and Alzheimer’s patients. Pretreatment of macrophage-conditioned media (MCM) with anti-cathepsin B antibodies or inhibitors reduces neuronal apoptosis and beta-amyloid peptides in vitro. We have demonstrated that recombinant active cathepsin B added in MCM is internalized by neurons, and the levels of internalization are proportional to the levels of HIV infection. Therefore, we hypothesize that targeting cathepsin B in MCM represents a viable approach to elucidate its mechanism of neuronal dysfunction. To test this hypothesis, we exposed SK-N-SH neuroblastoma cells to histidine-tagged cathepsin B in culture media alone or in presence of anticathepsin B antibody, and localized the histidine tag in neurons by intracellular flow cytometry. Histidine-tagged cathepsin B was internalized by neurons (52.0 %), a mechanism that was partially reduced by the pre-treatment with anti-cathepsin B antibody (34.9 %). The neuroprotective potential of cathepsin B antibody was confirmed by immunofluorescence, demonstrating increased neuronal MAP2 staining and recovered morphology. Then, we examined the presence of cathepsin B in exosomes released by uninfected and HIV-infected MDM at 12 days post-infection. Western blots revealed that exosomes isolated from MDM infected in vitro contain cathepsin B in both pro-peptide and mature forms, and express CD63, an exosome marker. Our results suggest that cathepsin B might be secreted in exosomes by HIV-infected macrophages, proposing a novel mechanism by which cathepsin B triggers neuronal dysfunction and opening doors for new studies in search for approaches to decrease the neuronal dysfunction and subsequent development of HAND. P21 A novel deep sequencing platform (ViroFind) reveals a complex JC virus population in the brain of PML patients Spyros Chalkias1, Joshua Gorham1, Erica Mazaika1, Michael Parfenov1, Xin Dang1, Steve DePalma1, David McKean1, Jonathan Seidman1, Igor Koralnik2 (corresponding author: ) 1Harvard Medical School; 2Rush University Medical Center Background: Deep sequencing enables the unbiased detection of viruses in clinical samples but it is limited by low costeffectiveness. We developed a novel target-enrichment deepsequencing platform (ViroFind) for the cost-effective sequencing of viral genomes and we investigated the composition of viral populations in the brain of 5 PML patients and in 18 controls with no known neurological disease. Methods: ViroFind comprises 165,433 probes, complementary to the entire genomes of 386 DNA and RNA viruses known to infect humans. The ViroFind probes are used in a hybridization reaction with nucleic acid libraries from clinical samples to enrich only viral sequences. The output of this reaction is sequenced via an Illumina platform and the sequencing data are aligned against a database of all viral genomes. Mapped sequences are used to analyze viral genomes and identify viral variants. Results: Compared to direct deep sequencing, ViroFind enriched the JC virus (JCV) sequences detected per PML brain sample, ranging from 584 viral sequences (each of 75 bp length) per 1,000,280 total sequences up to 375,653 viral sequences per 430,842 total sequences. The enrichment of JCV sequences attributable to ViroFind ranged from 33 to 127-fold. Each JCV nucleotide was sequenced at least 10 times (coverage 10X). We identified 24 viral capsid protein VP1 variants and 12 of these variants were associated with amino acid substitutions. Mutation D66H, likely related to VP1 conformational changes that could impact tissue tropism, was observed in 4 of 5 (80 %) PML brain samples. Lastly, we detected low frequency JCV sequences in 10 of 18 (56 %) control brains. Conclusion: Multiple JCV variants constitute highly complex viral populations in the brain of PML patients. JCV genome fragments can also be detected in the brain of up to 56 % of individuals with no known neurological disease, which suggests that JCV is present at a latent stage in the human brain. P22 HHV-6A infection induces the expression of Human Endogenous RetroViral elements (HERV-W-Env) in human cells: link to the neuroinflammation 1International Centre for Infectiology Research, INSERM U1111, CNRS UMR5308, Ecole Normale Superieure de Lyon, University of Lyon 1, 69365 Lyon, France; 2INSERM U823, Universite J Fourier, IAPC, 38041 Grenoble, France; 3GeNeuro Innovation, Lyon, France Herpesvirus infections have been associated with the induction of human endogenous retroviruses (HERV), suggesting they could synergistically participate in the establishment of different pathologies in humans. Among the HERV-W family, the envelope protein (HERV-W-ENV) seems to be involved in the pathogenesis of multiple sclerosis (MS). We have analyzed here the interaction between HHV-6A and HERV-W-ENV and have particularly focused on the transactivator role of CD46 molecule, acting as the receptor for several viruses, including HHV-6A and measles. We infected several primary and immortalized human cell lines, including T lymphocytes, monocytes, glioblastoma and neuroblastoma cells with HHV-6A (GS). The mRNA expression of ENV from different HERVs (syncytin-1, MSRV and HERV-K18) was determined by quantitative RT-PCR and results were confirmed by immunofluorescence. The involvement of CD46 in HERV-W-ENV activation was analyzed using several CD46 ligands, including UV-inactivated HHV-6A, measles virus and anti-CD46 antibody and recombinant C3b complement fragment, which binds naturally CD46. We showed that HERV-W-ENV over-expression in HHV-6A-infected human cells at transcript and protein level. Furthermore, the stimulation of HHV-6A receptor CD46, using UV-inactivated HHV-6A, anti-CD46-SCR3 domain Ab or C3b fragment of complement also increases the production of HERV-W-ENV, in contrast to measles virus, which binds different region of CD46 (SCR1-2 domains). These results suggested that induction of HERV-W is specific for HHV-6A infection in neural and immune cells and that engagement of CD46-SCR3 domain might be required for the induction of HERV-W. The reactivation of HERV-WENV was shown to induce inflammatory responses through TLR4. Therefore, these results expand the spectrum of HHV-6 effects and of its receptor CD46 in the modulation of the immune response and support the hypothesis that cross-talk between HHV-6A and HERV as well as complement activation and consequent CD46 activation may participate in the pathogenesis of the neuroinflammation. P23 Neuro-inflammation modulates Fcgamma receptor expression on microglial cells following viral brain infection Department of Medicine, University of Minnesota Medical School Fcgamma receptors (FcgammaRs) for IgG couple innate and adaptive immunity through their ability to activate effector cells by antigen-antibody complexes. Brain-resident microglial cells, which are pivotal to pathogen detection and initiation of innate neuro-immune responses, coexpress activating (FcgammaRI, FcgammaRIII, and FcgammaRIV) as well as inhibitory Fcgamma Receptor (FcgammaRIIB). Thus, the threshold for cellular activation is determined by the relative ratios of these opposing FcgammaRs. In this study, we investigated the relative expression of both activating and inhibitory FcgammaRs on microglia using an in vivo model of chronic neuroinflammation following murine cytomegalovirus (MCMV)induced encephalitis. Flow cytometric analysis of microglial cells obtained from infected brain tissue demonstrated that the activating FcgammaRs were expressed maximally at 5 d post infection (dpi), while the inhibitory receptor (FcgammaRIIB) remained highly elevated during the chronic phase of infection (i.e., 30 and 60 dpi). The highly induced expression of activating FcgammaRIV during the acute phase of infection was also noteworthy. While, FcgammaRI was found to be constitutively expressed, there was minimal expression of FcgammaRIII. Subsequently, when the relative expression of FcgammaRs was analyzed within the brains of mice post MCMV-infection by semiquantitative RT-PCR (semi-qRT-PCR), we observed maximal expression of FcgammaRIV during the acute phase commensurate with our flow c ytometric studies. Furthermore, in vitro analysis of cultured primary murine microglial cells using flow cytometry and semi-qRT-PCR demonstrated maximal expression of FcgammaRIV and FcgammaRIIB following stimulation with either IFNgamma or IL-4, respectively. We also demonstrated the role of IFN-gamma and IL-4 in polarizing microglia towards M1 or M2 phenotype, respectively. Thus, we conclude that acute neuro-inflammation following viral infection increases expression of activating FcgammaRs to promote pathogen clearance through increased effector cell activation. In contrast, preferential expression of the inhibitory receptor during chronic infection may provide a protective mechanism to prevent hyper-immune responses and subsequent bystander brain damage. P24 Computational detection of off-target effects of CRISPR/Cas9-associated gRNAs Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine Despite antiretroviral therapy, HIV-1 infection remains a life-long clinical problem due to reservoirs harboring proviral DNA in a latent/persistent form. Recently, gene-editing strategies utilizing the CRISPR/Cas9 system have been developed to eradicate the HIV-1 genome from infected cells. These results offer a new avenue toward the elimination of HIV-1 to cure HIV/AIDS. However, due to the promiscuous nature of the gRNA targeting, there is concern over offtarget binding sites that may cause unwanted DNA damage within the human genome that need to be assessed. This is complicated by the non-linear nature of the binding penalties associated with gRNA recognition. With the availability of unbiased identification of double stranded breaks enabled by sequencing (GUIDE-seq), the off-target cleavage sites can be precisely observed. In order to measure the offtarget effect, we have developed a new database containing all potential cleavage sites within the entire human genome along with all known single nucleotide polymorphisms in dbSNP. Tree construction occurs on the order of O(N) ~ Nlog(N) time where N is the size of the database and tree search occurs in O(m) ~ m where m is the length of the query. In practice, the entire human genome and dbSNP were indexed within 72 h and a query takes less than 10 ms per gRNA. This database will greatly increase the ability of researchers to design gRNA that can cleave HIV-1 while avoiding off-target effects. Future studies will be performed to validate the off-target predictions using biomedical research laboratory techniques including GUIDE-seq. P25 Altered mitochondrial biogenesis in brains during HAND: Another result of Tat-mediated neurotoxicity? 1Department of Psychiatry, University of California San Diego; 2Department of Neuroscience, University of California San Diego Background: Despite combined antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) remain a significant problem for the 1.4 million people living with HIV in the USA. The causes of HAND are probably a diverse set of initiating factors, such as low-level viral expression, drugs of abuse, cART neurotoxicity, a combination of these factors and others, which set in to action a cascade of inflammatory events that culminate in the neurodegeneration associated with HAND. Multiple recent publications implicate alterations in mitochondrial dynamics in neurons as a mechanism of neurotoxicity in some HAND patients. Methods: In the current study, we asked whether alterations in mitochondrial biogenesis activities might also contribute to the pathogenesis of HAND in the cART era. To this end, we analyzed expression levels and distribution patterns of mitochondrial biogenesis related genes and proteins in the frontal cortex of 37 well-studied cART-era HAND donors from the NNTC cohorts. Results: We found significant increases in the master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha, in brain tissues of HAND donors; mRNA levels also indicated altered PGC-1alpha expression. Immunohistochemichal analyses of PGC-1alpha show a distinct pattern in the brains of HAND donors compared to HIV- control. Importantly, we found similar alterations brains of in inducible Tat tg mice compared to their non-tg counterparts. Discussion: In combination with recent reports, our data suggest that alterations at both ends of the mitochondrial biogenesisdynamics axis may contribute to HAND, and Tat may be an important player in some patients. As mitochondrial dysfunction is implicated in many neurodegenerative Pascale Coric1, A. Sami Saribas2, Jason Saredy2, Martyn White2, Serge Bouaziz1, Mahmut Safak2 (corresponding author: ) Bianca Cotto1, William Yen1, Timothy Luongo2, Prasun Datta1, Satish Deshmane1, John Elrod2, Dianne Langford1 (corresponding author: ) disorders, further elucidation of these mechanisms may provide a promising therapeutic target. P26 Three Dimensional NMR Structure of the Human Polyomavirus JC Agnoprotein Revealed That the NH2and COOH-termini of the Protein Adopt Intrinsically Unstructured Conformation While the Center Portion Forms an Alpha-helix 1Universite Paris Descartes, Sorbonne Paris Cite, Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS, 4 av. de l’Observatoire, Paris, France; 2Department of Neuroscience, Center for NeuroVirology, Laboratory of Molecular Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, USA JC virus (JCV) causes a fatal demyelinating disease of the central nervous system (CNS), known as progressive multifocal leukoencephalopathy (PML), in a subset of the patients with underlying immunosuppression, including lymphoma, leukemia and AIDS due to the lytic infection of the glial cells, including oligodendrocytes, by the virus. The incidence of PML dramatically increased in the era of the AIDS epidemic and this disease has also been steadily increasing among autoimmune disorder patients, including multiple sclerosis and Crohn’s disease, who are treated with antibody-based drugs. JCV establishes a sub-clinical persistant infection in the body during early childhood, but upon reactivation, it causes the neurological manifestations of PML. JCV encodes a limited number of structural and regulatory proteins. Among those, agnoprotein has been shown to play essential regulatory roles during the viral replication cycle where JCV was demonstrated to be unable to sustain its productive life cycle in the absence of agnoprotein expression. Previous studies also demonstrated that agnoprotein forms highly stable dimers and oligomers through its Leu/Ile/Phe-rich domain. Subsequent NMR structural studies using a partial agnoprotein peptide revealed that the amino acids from Lys23 to Phe39, containing Leu/Ile/Phe-rich domain, adopt a alpha-helical conformation. Here we report the complete 3D structure of the full-length peptide of agnoprotein by NMR. This 3D structure confirmed the localization of the previously reported alpha-helical domain of the protein and further revealed the presence of another shorter alpha-helix located between residues Leu6 and Lys13. The remaining regions of the agnoprotein exhibit an intrinsically unstructured conformation, the significance of which has yet to be demonstrated. We are currently exploring the functions of these regions by mutagenesis. This study has been made poss i b l e b y g r a n t s a w a r d e d t o M . S a f a k f r o m N I H (1R01NS090949-01A1) and Temple University Drug Discovery Initiative (161398). P27 Effects of cocaine and Tat on astrocyte-derived cholesterol supplied to neurons 1Department of Neuroscience, Center for Neurovirology; 2Department of Pharmacology, Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, PA USA The human brain requires a balance of cholesterol homeostasis to meet the high metabolic requirements of neurons and maintain myelin integrity. Neurons rely on cholesterol synthesized by astrocytes for synaptogenesis and normal functioning. In the brain, cholesterol synthesis and clearance are tightly regulated to maintain cholesterol levels. Liver X receptors (LXRs) are the master regulators of cholesterol homeostasis in the CNS. LXR activation leads to the transcription of target genes involved in cholesterol trafficking and efflux, including apolipoprotein E (ApoE). The disturbance of LXR signaling in the brain can lead to significant dysfunction in cholesterol homeostasis and disruptions have been implicated in several neurological diseases. Likewise, HIV infection and cocaine use are associated with myelin loss and synaptodendritic damage suggesting that dysregulation in CNS cholesterol metabolism may be involved in the progression of neurological impairment. Therefore, given the importance of astrocytes in LXR-mediated cholesterol regulation and its role in providing metabolic support to other CNS cells, we hypothesized that exposure of astrocytes to cocaine and Tat would lead to disruptions in LXR signaling and the expression of LXR regulated genes. Alterations in these pathways may in turn decrease the bioavailability of cholesterol from astrocytes to neurons and oligodendrocytes and promote cellular dysfunction. We found that in astrocytes, exposure to cocaine and Tat significantly decreased LXRbeta expression. In addition, we discovered that LXR target genes including ApoE were also significantly reduced upon treatment with cocaine and Tat. In response, the ABC1a transporter responsible for transporting ApoE bound cholesterol out of the cell is increased significantly. Our findings demonstrate the combined effects of cocaine and Tat on the overall bioavailability of cholesterol provided by astrocytes to neurons and identify a novel role for LXR agonist as a therapeutic intervention in the severity and progression of HIV infection and/or cocaine abusing individuals. P28 Microglial activation and cocaine addiction Bianca Cotto1, Christina Chambers2, Hongbo Li2, William Yen1, Ronald Tuma2, Sara Jane Ward2, Dianne Langford1 (corresponding author: ) 1Department of Neuroscience, Center for Neurovirology; 2Department of Pharmacology, Center for Substance Abuse Research, Lewis Katz School of Medicine at Temple University, Philadelphia, PA USA Recent research shows that glial cells are intimately involved in synaptic plasticity, and that drugs of abuse affect glial activity. Specifically, microglia contribute to synaptic plasticity via direct interactions with dendritic spines, synaptic pruning, and regulation of hippocampal neurogenesis. We have combined a series of in vivo, ex vivo, and in vitro experiments to test the hypothesis that cocaine exposure can lead to significant alterations in microglial activation, enhancing cocaine-induced neuroplasticity. In turn, these cascades contribute to peripheral immune cell invasion, thereby priming the neuroimmune axis for a cycle of neuroinflammation in the context of cocaine addiction. We found that cocaine self-administration in the rat model increases microglial activation in reward regions of the brain, and increases expression of the transcription factor and synaptic plasticity marker MeCP2. We then determined in vitro that microglia express MeCP2 and that this expression increases significantly following cocaine exposure and inducing release of BDNF, suggesting that cocaine’s effects on MeCP2 expression may participate in cocaineinduced neuroplasticity. In addition, we observed that chronic cocaine administration increases cerebrovascular leukocyte rolling and adhesion and subsequent BBB weakening that persists during withdrawal, setting up the likelihood f or a persistent dysregulation of neuroimmune signaling that may mirror the cycle of cocaine addiction. These findings identify novel neuroimmune targets such as activated microglia for treating psychostimulant abuse, for which no approved medications exist. Our findings also illustrate mechanisms in the cocaine abuse paradigm that are likely important in CNS disease progression in HIV-infected patients who use cocaine. P29 Agnoprotein interferes with the neuroimmune response to JC Virus Michael Craigie, Stephanie Cicalese, Martina Donadoni, Ilker Sariyer (corresponding author: ) Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA USA JC virus is a human neurotropic polyomavirus and the e t i o l o g i c a g e n t o f p r o g r e s s i v e m u l t i f o c a l leukoencephalopathy, a demyelinating disease of gray and white matter. PML is primarily observed in immunocompromised patients, including patients with AIDS and those undergoing immunomodulatory therapies. During JCV infection, the virus encodes multiple viral proteins including the regulatory agnoprotein. We have recently discovered that agnoprotein is secreted from infected cells into the extracellular matrix. Also, previous data demonstrated that JCV infection is suppressed by treatment with conditioned media from peripheral blood mononuclear cells, suggesting that the immune system and immunological soluble factors play a major role in the regulation of JCV. Here, we have investigated the possible role of extracellular agnoprotein in the neuroimmune response to JCV in glial cells. Following the cellular expression of agnoprotein, agnoprotein is released by glial cells and is able to be internalized by both astrocytes and microglia in vitro. This internalization of agnoprotein was found to impact the profile of cytokines released by both cell types. Moreover, the treatment of astrocytes with media containing agnoprotein resulted in a significant reduction in GM-CSF secretion. Subsequent reporter gene analysis demonstrated that agnoprotein is capable of suppressing the transcription of GM-CSF, implicating a possible mechanism for the reduction of released GM-CSF levels. Likewise, the treatment of a human monocyte cell line, U937, with agnoprotein resulted in decreased macrophage differentiation, including decreased attachment and decreased phagocytic ability. Similarly, treatment of peripheral blood mononuclear cells with agnoprotein resulted in decreased overall migration across an in vitro blood brain barrier model. These findings have suggested that extracellular agnoprotein is able to modulate aspects of the immune response to JCV, primarily through the suppression of GM-CSF release and subsequent impact on monocyte/ macrophage function. P30 HIV-1 genetic variation resulting in the development of new quasispecies continues to be encountered in the peripheral blood of well-suppressed patients William Dampier1, Michael Nonnemacher1, Joshua Mell1, Joshua Earl1, Vanessa Pirrone1, Benjamas Aiamkitsumrit1, Wen Zhong1, Katherine Kercher1, Shendra Passic1, Jean Williams1, Zsofia Szep2, Jeffrey Jacobson3, Brian Wigdahl1 (corresponding author: ) 1Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease; 2Department of Medicine, Division of Infectious Diseases and HIV Medicine, Drexel University College of Medicine; 3Department of Medicine, Section of Infectious Disease, Lewis Katz School of Medicine, Temple University As a result of antiretroviral therapeutic strategies, human immunodeficiency virus type 1 (HIV-1) infection has become a long-term clinically manageable chronic disease for many infected individuals. However, despite this progress in therapeutic control, including undetectable viral loads and CD4+ T-cell counts in the normal range, viral mutations continue to accumulate in the peripheral blood compartment over time, indicating either low level reactivation and/or replication. Using patients from the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort, whom have been sampled longitudinally for more than 7 years, genetic change was modeled against the dominant integrated proviral quasispecies with respect to selection pressures such as therapeutic interventions, AIDS-defining illnesses, and other factors. Phylogenetic methods based on the sequences of the LTR and tat exo n 1 of the HIV-1 proviral DNA quasispecies were used to obtain an estimate of an average mutation rate of 5.3 nucleotides (nt)/kilobasepair (kb)/ year (yr) prior to initiation of antiretroviral therapy (ART). Following ART the baseline mutation rate was reduced to an average of 1.02 nt/kb/year. The post-ART baseline rate of genetic change, however, appears to be unique for each patient. These studies represent our initial steps in quantifying rates of genetic change among HIV-1 quasispecies using longitudinally sampled sequences from patients at different stages of disease both before and after initiation of combination ART. Notably, while long-term ART reduced the estimated mutation rates in the vast majority of patients studied, there was still measurable HIV-1 mutation even in patients with no detectable virus by standard quantitative assays. Determining the factors that affect HIV-1 mutation rates in the peripheral blood may lead to elucidation of the mechanisms associated with changes in HIV-1 disease severity. P31 Defining Vpr sequence variants and associated mechanisms that enhance HIV CNS pathogenesis and disease William Dampier1, Gregory Antell2, Michael Cruz3, Benjamas Aiamkitsumrit1, Michael Nonnemacher1, Zsofia Szep4, Jeffrey Jacobson5, Vanessa Pirrone1, Wen Zhong1, Katherine Kercher1, Shendra Passic1, Jean Williams1, Tony James1, Kathryn Devlin6, Tania Giovannetti7, David Libon8, Garth Ehrlich1, Brian Wigdahl1, Fred Krebs1 (corresponding author: ) 1Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine; 2School of Biomedical Engineering, Science, and Health Systems, Drexel University; 3Interdisciplinary Health Sciences (IHS) Program, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine; 4Department of Medicine, Division of Infectious Diseases and HIV Medicine, Drexel University College of Medicine; 5Department of Medicine, Section of Infectious Disease, Lewis Katz School of Medicine, Temple U n i v e r s i t y ; 6 D e p a r t m e n t o f N e u r o s c i e n c e a n d Comprehensive NeuroAIDS Center, Lewis Katz School of Medicine, Temple University; 7Department of Psychology, Temple University; 8Department of Geriatrics and Gerontology, New Jersey Institute for Successful Aging, School of Osteopathic Medicine, Rowan University Studies of HIV-associated neurocognitive disorders (HAND) have identified viral protein R (Vpr) as a neuropathogenic factor. Extracellular Vpr, which has been detected in the sera and cerebrospinal fluids of HIV-1infected patients, causes adverse changes in brain resident cells, including macrophages, astrocytes, and neurons. Our recent studies of Vpr have turned to investigating connections between naturally occurring Vpr sequence variation and neuropathogenesis in HIV-1-infected patients from the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. Analyses of peripheral bloodderived Vpr sequences from Cohort patients revealed three amino acids in Vpr (positions 37, 41, and 55 in the 96 aa protein) associated with significant differences in neuropsychological impairment (niVpr variants) measured by comprehensive neuropsychological testing. 37-I and 41-S were linked to reductions in diagnosed neuropathogenesis (with respect to the average Global Deficit Score or GDS), while 41-N and 55-A were associated with increased neuropathogenesis and higher GDS. The effects of niVpr variants appear to be cumulative, since the combined effect sizes for Vpr sequences with two or three niVpr variants preliminarily correlate with changes in average patient GDS. Deep sequencing demonstrated the appearance of niVpr variants in HIV-1-infected patient blood, spleen, and brain samples, and indicated sequence compartmentalization as well as variations in relative prevalence. The pathogenic effects of Vpr variation on immunopathogenesis and neuropathogenesis were suggested, respectively, by higher CD4 T-cell counts in patients with the 41-N variant (relative to cell counts in patients with the 41-S variant) and greater reductions in astrocyte glutathione pools subsequent to transient expression of the 41-N Vpr variant (relative to the 41-S variant). P32 Astrocyte-shed extracellular vesicles regulate synchronous network burst activity 1Department of Neurology, Johns Hopkins University School of Medicine; 2Department of Neurology, Johns Hopkins University School of Medicine. Department of Psychiatry, Johns Hopkins University School of Medicine Synaptic damage and disruption of neuronal networks are prominent features of HIV Associated Neurocognitive Disorders (HAND). The underlying mechanisms for these pathological alterations in synaptodendritic networks are largely unknown, but are thought to involve chronic glial activation and inflammation. Based on evidence that astroglia to neuron communication regulates the formation and strength of synaptic connections, we hypothesized that HIV infection disrupts neural network activity by modifying the quality of astrocyte to neuron communication. Extracellular vesicles (EVs) are emerging as a mechanism by which astroglia regulate neuronal communication through the delivery of protein, RNA and lipid cargo that regulate the activity of target cells. In this study we isolated exosomes shed from astrocytes in response to ATP (a trophic stimulus), or media from HIV-infected macrophages (HIV-MDM; HIV and EV depleted), and exposed primary rodent neurons to a dose response of 5–100 EVs/cell. The effects of EVs on dendritic length, branching, and complexity were determined using structured fluorescent imaging, and neural connectivity was determined using multichannel electrode arrays. Spike detection was accomplished using filtered voltage measurements with a threshold for detection of 5 standard deviations above baseline. A MATLAB based code was developed to extract spike times that allow for the creation of 3D animations to visualize spike progression in individual electrodes over time, burst identification, spike and burst firing rates, and periodicity detection. We found that exosomes released from astrocytes in response to ATP stimulation produced a dose-dependent increase in dendritic complexity, increased neural network connectivity, and periodic synchronous network burst activity. In contrast, exosomes shed from astrocytes in response to HIV-MDS reduced dendritic complexity. These data suggest that EVs shed from astrocytes strengthen neural network connectivity and in the setting of HIV infection EVs may deconstruct neural networks possibly by modifying the composition of EV cargo. P33 Follicular DCs within deep cerebral lymph nodes are potential reservoir of HIV-CNS infection 1Department of Microbiology and Immunology, and the Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA; 2Division of Infectious Disease and HIV Medicine, Department of Medicine, Drexel University College of Medicine, Philadelphia, PA; 3AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD; 4Division of Infectious Disease and HIV Medicine, Department of Medicine, Drexel University College of Medicine, Philadelphia, PA; 5Department of Pathology & Laboratory Medicine, School of Medicine and Emory Va c c i n e C e n t e r, E m o r y U n i v e r s i t y, A t l a n t a , G A ; 6 D e p a r t m e n t o f P h a r m a c o l o g y a n d E x p e r i m e n t a l Neuroscience, University of Nebraska Medical Center, Omaha, NE Human immunodeficiency Virus (HIV) neuroinvasion leads to neurodegeneration and HIV-associated neurocognitive disorder. In the central nervous system (CNS) circulating dendritic cells (cDCs) encounter HIV virions or viral proteins and migrate along the rostral migratory system to the cervical or cerebral lymph nodes (CxLNs). Subsequently, HIV accumulates in the germinal centers (GCs) and there it appears bound to follicular dendritic cells (FDCs). In this study, we sought to dissect the mechanisms underlying harboring of HIVon FDCs and their transmission to CD4+ T follicular helper (TFH) cells in CxLNs of chronically SIV-infected rhesus macaques (RMs). Chronic SIV infection was established in all RMs. CD35+ FDCs in CxLNs were identified to harbor SIV. Furthermore, FcRs (FcgammaRIIB) immune complexes colocalized with the FDCs and B-cells, suggesting SIV presentation by FDCs to B-cells. Significantly, B-cell interaction with TFH cells resulted in the latter getting infected. Hence, FDCs in CxLN not only retained SIV, but they also infected TFH cells. Based on these observations, we infer a novel role for FDCs in viral entrapment and creation of persistent CNS reservoir. Hence, our study highlights the role of FDCs in accumulation and transmission of HIV with implications for HIV egress from the CNS. P34 Sex Matters: Differential Deficits in Sustained Attention in the Male and Female HIV-1 Transgenic (Tg) Rat Iris K. Dayton, Kristen A. McLaurin, Rosemarie M. Booze, Charles F. Mactutus (corresponding author: ) Department of Psychology, University of South Carolina Neurocognitive impairment (NCI) is a prevalent condition of HIV-1 infected individuals because of the remarkable success of combined antiretroviral therapy. Recent studies suggest an effect of gender on the domains of speed of information processing, verbal fluency, learning, and memory. Although it has been reported that the risk for developing NCI for HIV-1 men and women differs, the examination of biological sex differences in NCI, independent of societal factors, has not been previously conducted. Deficits in executive function are a distinctive characteristic of NCI. Presently, we examined the potential sex differences in sustained attention deficits, as a core component of executive function using the HIV-1 Tg rat. Male Fischer HIV-1 Tg (n = 35) and F344/N control rats (n = 35), and intact female Fischer HIV-1 Tg (n = 35) and F344/N control rats (n = 33) were trained (shaping) and tested (vigilance and manipulation of stimulus duration) in a signal detection task. There were marked sex differences in initial training of the signal detection task, acquisition of the vigilance task, and performance on the duration task. Females acquired the signal detection task at a much slower (4X) rate than males. Females met criterion of five consecutive days or seven non-consecutive days at 70 % or higher for the vigilance task at a significantly slower rate than males; there was a 14-day lag before any female met the acquisition criterion. The factor of biological sex altered the ability to discriminate signals (illumination for 100, 500, or 1000 msec duration) from non-signals (no illumination) during the duration task. Both HIV-1 Tg and control females were unable to discriminate signals presented at shorter durations than their male counterparts. Collectively, these findings suggest that the factor of biological sex has a significant effect on the development of NCI for HIV-1 patients. Funded by NIH grants DA013137, HD043680, and MH106392. P35 IFN-Gamma Inhibits JC Virus Replication in Glial Cells by Suppressing T-Antigen Expression. Francesca Isabella De Simone, Rahsan Sariyer, Shadan Yarandi, Michael Craigie, Jennifer Gordon, Ilker Sariyer (corresponding author: ) Temple University Lewis Katz School of Medicine Patients undergoing immune modulatory therapies for the treatment of autoimmune diseases such as multiple sclerosis, and individuals with an impaired-immune system, most notably AIDS patients, are in the high risk group of developing progressive multifocal leukoencephalopathy (PML), an often lethal disease of the brain characterized by lytic infection of oligodendrocytes in the central nervous system (CNS) with JC virus (JCV). Here, we investigated the impact of soluble immune mediators secreted by activated PBMCs on viral replication and gene expression by cell culture models and molecular virology techniques. Our data revealed that viral gene expression and viral replication were suppressed by soluble immune mediators. Further studies demonstrated that soluble immune mediators secreted by activated PBMCs inhibit viral replication induced by T-antigen, the major viral regulatory protein, by suppressing its expression in glial cells. This unexpected suppression of T-antigen was mainly associated with the suppression of translational initiation. Cytokine/ chemokine array studies using conditioned media from activated PBMCs revealed several candidate cytokines with possible roles in this regulation. Among them, only IFN-gamma showed a robust inhibition of T-antigen expression. While potential roles for IFN-beta, and to a lesser extent IFN-alpha have been described for JCV, IFN-gamma has not been previously implicated. Further analysis of IFN-gamma signaling pathway revealed a novel role of Jak1 signaling in control of viral T-antigen expression. Furthermore, IFN-gamma suppressed JCV replication and viral propagation in primary human fetal glial cells, and showed a strong anti-JCV activity. Our results suggest a novel role for IFN-gamma in the regulation of JCV gene expression via downregulation of the major viral regulatory protein, T-antigen, and provide a new avenue of research to understand molecular mechanisms for downregulation of viral reactivation that may lead to development of novel strategies for the treatment of PML. play a crucial role in neurotoxicity associated with alcohol consumption. P36 Alcohol-Mediated Alternative Splicing of Mcl-1 pre-mRNA is Involved in Ethanol Induced Neurotoxicity P37 The role of infiltrating macrophages and IL-6 production on seizure development after viral infection Francesca Isabella De Simone1, Rahsan Sariyer1, Sulie L. Chang2, Ilker Sariyer1 (corresponding author: ) Ana Beatriz DePaula-Silva, Tyler Hanak, Daniel Doty, Jordan Sim, Jane Libbey, Robert Fujinami (corresponding author: ) 1Department of Neuroscience, Center for NeuroVirology, Lewis Katz School of Medicine at Temple University; 2Institute of NeuroImmune Pharmacology, Seton Hall University Heavy and chronic ethanol exposure can cause significant structural and functional damage to the adult brain. The developing nervous system is even more vulnerable to ethanol exposure. Prenatal exposure of ethanol during pregnancy can lead to fetal alcohol spectrum disorders (FASD), characterized by malformation of the nervous system and mental retardation. The most devastating consequence of ethanol exposure is the neurotoxicity associated with the depletion of neurons. It is crucial to elucidate mechanisms of neuroapoptosis in order to develop effective therapeutic approaches to overcome ethanol-induced neuropathologies. Regulation of splice variants in the brain can modulate protein functions, which may ultimately affect behaviors associated with alcohol dependence and ethanol-mediated neurotoxicity. Limited number of studies has shown that pre-mRNA splicing patterns of genes are potentially altered and involved in behavior changes associated with alcoholism. Since alcohol consumption is associated with neurotoxicity, it is possible that altered splicing of survival and pro-survival factors during the development of alcoholism may contribute to the neurotoxicity. Our results suggest that ethanol exposure can lead to premRNA missplicing of Mcl-1, a pro-survival member of the Bcl-2 family, by downregulating the expression levels of serine/arginine rich splicing factor 1 (SRSF1). The pre-mRNA of Mcl-1 can be alternatively spliced to remove exon 2, which produces shortened form of Mcl1, named Mcl-1S. While the longer gene product Mcl1 L enhances cell survival, the alternatively spliced shorter gene product Mcl-1S promotes apoptosis. Our preliminary data has indicated that ethanol exposure to neurons leads to a decrease in the ratio of Mcl-1 L/Mcl1S by favoring pro-apoptotic Mcl-1S splicing over antiapoptotic Mcl-1 L isoform suggesting that Mcl-1S may Epilepsy is a chronic neurological disorder characterized by recurrent seizures. Seizures, which are often associated with viral encephalitis, occur in response to imbalances between excitatory and inhibitory inputs within the brain. Patients with viral encephalitis are 16 times more likely to develop epilepsy. It is estimated that around 65 million people worldwide suffer from epilepsy. Although treatments to prevent seizures are available, they are mainly anticonvulsants and around 30 % of the patients do not respond to the medication and numerous side effects related to the drugs have been reported. In order to develop new immunomodulatory treatments that could lead to an eventual cure for seizures/epilepsy, a better understanding of the seizure development mechanism is required. We have developed an experimental model of virus-induced seizures/epilepsy. In our model, C57BL/6 mice are infected intracranially (i.c) with the Daniels strain (DAV) of Theiler’s murine encephalomyelitis virus (TMEV). Between 3 and 10 days post infection (d.p.i), around 50 % of the infected mice will develop spontaneous acute seizures and approximately 50 % of the mice that have experienced spontaneous acute seizures will develop epilepsy. Because seizures begin to develop at 3 d.p.i, we believe it happens as a consequence of the activation of the innate immune response. We found that seizures are correlated with an increase in myeloid and lymphoid cells infiltrating into the brain. We also found that the pro-inflammatory cytokines IL-6 and TNF alpha were elevated at 3 d.p.i. IL-6 knockout mice infected with DAV experienced significant fewer seizures. Because infiltrating macrophages are the main producers of IL-6, we depleted macrophages from mice and we infected them with DAV. We did not observed seizures in animals lacking macrophages, suggesting macrophages are playing a central role in the development of seizures after viral infection. P38 Macrophage OXPHOS regulation by HIV-1 Vpr and cocaine Satish Deshmane, Shohreh Amini, Prasun Datta (corresponding author: ) Neuroscience, Comprehensive NeuroAIDS center, LKSOM at Temple University HIV-1 infected macrophages play a significant role in the pathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. Mitochondria are key players in the generation and regulation of cellular bioenergetics, producing the majority of adenosine triphosphate molecules by the oxidative phosphorylation system (OXPHOS). We therefore assessed the effects of Vpr alone and Vpr in combination with cocaine on the relative protein expression of OXPHOS mitochondrial complexes in macrophages. U937 differentiated macrophages were transduced with adenoviral vector harboring Vpr gene. Transduced cultures were either left untreated or treated with 5‘microM of cocaine for 1 and 3 days. Cell-lysates were prepared and electron transport complexes were detected using an antibody mix that detects key component proteins from each complex (CI-NDUFB8, CII-SDHB, CIII-UQCRC2, CIV-MTCO1 and CV-APT5A). Complex III, IV and V levels stayed constant however, complex I and II were found to be negatively affected by Vpr. Complex-I was found to be downregulated by 32 % on day 1 and day 3 post-transduction with Vpr. Complex-II was found to be downregulated by 33 and 71 % on day 1 and day 3. With cocaine, a transient upregulation of complex I and II was found on day 1. However, both complexes I and II were found to be downregulated by 70 % and 32 % respectively on day 3 post-transduction. Together, our data show that Vpr and cocaine reprogram macrophage OXPHOS. Funded by NIDA. (corresponding author: ) 1Section of Infection of the Nervous System; National Institute of Neurological Disease and Stroke (NINDS); NIH; 2Bioinformatics Section of Information Technology Program, NINDS, NIH; 3Neural Differentiation Unit, NINDS, NIH; 4 C o m m u n i t y N e t w o r k s P r o g r a m , S e c t i o n o f Neurophysiology and Biophysics, NINDS, NIH; 5National Cancer Institute, NIH; 6Section of Infection of the Nervous System, NINDS, NIH Human Endogenous Retroviruses (HERVs) comprise approximately 8 % of the human genome and play a critical, highly regulated spatiotemporal role in cellular differentiation during development. We hypothesize that HERVs play a critical role in neuronal differentiation, and their dysregulation results in neurodevelopmental tumors. Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare type of pediatric brain cancer. The tumor cells have an expression profile similar to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We characterized the cell line by immunostaining and found the cells strongly express Pax6, a neuro-ectodermal marker, and Oct4, an iPSC marker while having low expression of Beta tubulin, a neuronal marker, and Nestin, a neuronal stem cell marker. Interestingly, when the AT/RT cell line was grown as an adherent line using matrigel and incubated in differentiation media, the cells began to differentiate into neuronal precursors and neurons with increased expression of both MAP2 and Nestin proteins. We analyzed each of the cell types and determined the stage of differentiation using RT-PCR and qPCR; furthermore, we compared the AT/RT derived neuronal cells with neurons generated from iPSCs. Similar to iPSCs, the AT/RT cells expressed HERV-K; however, HERV-K expression decreased dramatically as the cells differentiated into neuronal precursors and finally neurons. When we blocked HERV-K expression using siRNA in iPSCs we observed neuronal differentiation as determined by RT-PCR, qPCR, and immunostaining. Our data leads us to conclude that HERV elements likely play a key role in neuronal differentiation; furthermore, it raises the possibility that manipulation of HERV expression could be utilized as a treatment modality for neurodevelopmental tumors. P39 Critical Role of Human Endogenous Retroviruses (HERVs) in Neurodevelopment and Neurodevelopmental Tumors. P40 Use of a lip scarification mouse model to study the pathology, immunology, and virology associated HSV-1 Infection of the periphery and corresponding trigeminal ganglia Kevin Egan, Alex Allen, Brian Wigdahl, Stephen Jennings (corresponding author: ) Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine Herpes simplex virus type 1 (HSV-1) replicates in epithelial cells of mucosal surfaces before establishing a lifelong latent infection within the trigeminal ganglion. Although there is an established ocular infection model in the laboratory mouse which reproduces primary infection and latency observed in human, the majority of primary human infections occur within the lip and latency is established within a different branch of the trigeminal ganglion. Based on this, the lip scarification model has been used to study the genetic loci of HSV-1 resistance in the laboratory mouse, however the basic kinetics of and the pathologic and immunologic responses to viral infection have not been fully defined. Using the McKrae strain of HSV-1, we have proceeded to define the kinetics of viral infection in the lip. The lower lip of 3-month-old mice were scarified and inoculated with HSV-1 and tissue was collected at 7 time points up to day 60 post infection for detection of infectious virus and responding immune cells. High virus titers were detected in the lip at early time points that resolved after 8 days of exposure. Lip pathology peaked 5 days post-infection and resolved 15 days post-infection with residual lymphocytes present. CD45+ cells including CD4+ and CD8+ T lymphocytes infiltrated the TG and associated with neurons. CD8+ and CD4+ T lymphocytes were retained in the trigeminal ganglia and were present at 30 and 60 days post-infection. Analysis of genome copies revealed that genome copies peaked at 8 days post-infection in the lip. In the trigeminal ganglia genome copies established a steady level at 5 days post-infection. These results are the first descriptions of HSV-1 kinetics and pathology in the mouse lip. This model will be useful in studying the virologic and immunologic aspects associated with HSV-1 infection of the lower lip where most human infections occur. P41 Novel molecular pathway of perturbation of ATP release in astrocytes contributes to neuronal death in HAND 1Cellular and Molecular Neuroscience, National Brain Research Centre, Manesar, India; 2Cellular and Molecular Neuroscience, National Brain Research Centre, Manesar Astroglia are indispensable component of the tripartite synapse that regulate neuronal functions, health and survival through dynamic neuroglia crosstalk. Susceptibility to HIV-1 infection and subsequent latency in astrocytes contribute to neuronal damage that culminates into HIV-1 Associated Neurocognitive Disorders (HAND). The neurotoxic viral protein HIV-1 Transactivator of Transcription (Tat), is released in the brain even in successful cART cases. Tat is reported to be produced in HIV-1 infected astrocytes. Recently, we have demonstrated that astrocytes mediate indirect neuronal death by releasing excess ATP through activation of purinergic receptor system and experimentally proved that enhanced ATP levels induce astrocytes to secrete more MCP-1(CCL2), that is critical for monocyte aided viral trafficking into CNS. Using well characterized model system of human primary astrocytes and neurons, we further probed into the molecular mechanism for enhanced ATP release in Tat affected astrocytes. It was observed that HIV-1 Tat modulates the miRNA machinery in astrocytes to perturb the levels of voltage dependent anion channel-1 (VDAC1), a channel present in the outer mitochondrial membrane and plasma membrane which regulates extracellular ATP release. We report a novel molecular cascade of miRNA-mediated ATP release through regulation of VDAC1 and provide evidence that HIV-1 Tat dysregulates miR- 320a that in turn perturbs astrocytic ATP release. Down-regulation of VDAC1 either with miR-320a mimic or VDAC1 siRNA in HIV-1 Tat affected astroglia, could rescue the neurons from glia-mediated indirect death. We corroborated these in vitro findings using HIV-1 patients suffering from mild cognitive disorder (MCI) and age-matched control; we observed dysregulated miR-320a- VDAC1 axis in frontal cortex of HIV-MCI patients as compared to control subjects. Our findings reveal a novel upstream therapeutic target that could be employed to abolish the astroglia-mediated neurotoxicity in HIV neuropathogenesis. P42 Expression of mitochondrial biogenesis and innate inflammation genes in brains of HIV+ donors stratified by mtDNA haplogroups Jerel Fields, Hamza Coban, Sarah Gough, Ilse Flores, Eliezer Masliah, Paula Desplats, Cristian Achim (corresponding author: ) University of California, San Diego Despite combined antiretroviral therapy (cART), HIVassociated neurocognitive disorders (HAND) remain a significant problem for 1.4 million people living with HIV in the USA, and minority populations are disproportionately affected. The causes of HAND are multifactorial and may include low-level viral expression, drugs of abuse, cART longer-term or chronic cocaine treatment MSK1 is the main facilitator of HIV1 transcription. These activation events enhance the interaction of NF-kappaB with histone acetyltransferases (HATs) and promote the recruitment of the positive transcription elongation factor b (P-TEFb) to the H I V- 1 LT R , s u p p o r t i n g t h e d e v e l o p m e n t o f a n open/relaxed chromatin configuration, and facilitating both the initiation and elongation phases of HIV-1 transcription. Results are also confirmed in primary monocyte derived macrophages (MDM). Overall, our study provides detailed insights into cocaine-driven HIV-1 transcription and replication. P139 HIV-1/gp120 protein and methamphetamine induce alterations in dopaminergic and serotonergic neurotransmission systems 1Sanford Burnham Prebys Medical Discovery Institute, Infectious and Inflammatory Disease Center; 2UCSD, Translational Methamphetamine AIDS Res. Ctr. Individuals infected with human immunodeficiency virus type-1 (HIV-1) frequently use methamphetamine (METH). The incidence of both HIV-1 gp120 viral protein and METH in the central nervous system (CNS) is suspected to exacerbate HIV-associated neurocognitive disorders (HAND). In addition METH is a psychostimulant drug that compromises several neurotransmitter systems, including the dopaminergic and serotonergic network. However, the combined effects of HIV1 and METH on the brain are incompletely understood at the molecular level. We recently treated 3–4 months old HIV-1/ gp120 transgenic (gp120tg) and wild type (wt) mice with an escalating METH binge regimen for 25 days. At 10– 12 months of age, HIV-1/gp120tg and METH-exposed animals showed significant impairment in spatial learning and m e m o r y a n d n e u r o p a t h o l o g y. M E T H - e x p o s e d a n d HIVgp120tg animals also displayed changes components of the glutamatergic and GABAergic neurotransmission systems. METH-treated HIVgp120tg mice were the most severely affected. In order to further investigate underlying mechanisms in the brain, we used in the present study RT-qPCR arrays to assess expression of genes related to the dopaminergic and serotonergic neurotransmission systems. Six comparisons between the four experimental groups revealed significant gene regulation due to METH exposure and chronic HIV1/gp120 expression: 1) WT Saline (SAL) versus (vs.) WT METH: e.g. BDNF, AKT1, VMAT2, SERT1; 2) WT SAL vs. gp120 SAL: e.g. GFAP, HTR6, SYNPHILIN, CDK5; 3) WT SAL vs. gp120 METH: e.g. GFAP, BDNF, DRD5, CASP3, HTR1D/7; 4) WT METH vs. gp120 SAL: e.g. GFAP, HTR6, CDK5; 5) WT METH vs. gp120 METH: e.g. GFAP, B2M, HTR1A/1D/1 F/2C/4/7, VMAT1; 6) gp120 SAL vs. gp120 METH: e.g. GFAP, DRD5, HTR1A/1D/2C/4/7. In summary, histopathology and impaired spatial learning and memory due to METH exposure and HIV-1 gp120 expression are associated with significant alterations in the four major neurotransmission systems of the brain. P140 Role of autophagy and extracellular vesicles in neurotoxicity associated with HIV-1 Nef Sami Saribas, Stephanie Cicalese, Taha Ahooye, Kamel Khalili, Shohreh Amini, Ilker Sariyer (corresponding author: ) Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, PA HIV-associated neurological disorders (HAND) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer due to combined anti-retroviral therapies (cART). The virus utilizes viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among these viral proteins, Nef is a small HIV-1 protein expressed abundantly in astrocytes of HIV-1infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the CNS, Nef was expressed in primary human fetal astrocytes (PHFA) using an adenovirus. Our results revealed that Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, Nef release in EVs was enriched significantly when the cells were treated with perifosine, MG-132, LY294002, and wortmannin suggesting a novel role of autophagic signaling in Nef release from astrocytes. Next, Nef-carrying exosomes were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that Nef containing extracellular vesicles were readily taken up by neurons as evidenced by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef -carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed Nef in primary neurons by adenoviral transduction. Intracellular expression of Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nefcarrying exosomes suppressed functional neuronal firing activity assessed by multi-electrode array studies (MEA). Collectively, these data suggested that HIV-1 Nef can be a formidable contributor to neurotoxicity along with other factors which leads to HAND in HIV-1 infected AIDS patients. P141 Discovery and Characterization of the Novel ORFs Resulting from Trans-Splicing of the Human Polyomavirus JC Late Transcripts A. Sami Saribas, Jason Saredy, Martyn White, Mahmut Safak (corresponding author: ) Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, USA RNA splicing is a highly regulated nuclear processing event where various combinations of exons from premRNA molecules are jointed together to generate multiple protein isoforms by cis-and trans-splicing. The JC virus genome is known to transcribe two primary transcripts from its early and late coding regions and produces several predicted alternatively spliced products by cis-splicing, capable of encoding regulatory and structural proteins. Our recent NMR-based mutational analysis of the agnoprotein dimerization domain transcripts by RT-PCR has led us discover two new open reading frames (ORF1 and ORF2) by trans-splicing. These unexpected ORFs results from (i) a trans-splicing of the 5’short coding region of VP1 between the coding regions of both agnoprotein and VP2 after replacing the intron 1 and (ii) a further frame-shift occurring within the 5’-short coding sequences of VP2. ORF1 and ORF2 have the capacity to generate 58 and 72 aa long proteins respectively. Initial characterization of these ORFs by RT-PCR utilizing total RNA isolated from both the infected primary tissue culture cells and the PML brain tissue samples has confirmed that both ORFs are transcribed not only in vitro but also in vivo. Mutagenesis analysis of the ORF1 in the viral background suggests that it may play a role in transport of viral capsids into nucleus. Subsequent analysis of the possible protein coding capacity of the ORF1 by immunoblotting studies using newly raised polyclonal antibodies against its predicted amino acid coding sequence has revealed that it does indeed encode a small protein. Immunocytochemistry studies revealed that both ORFs mostly localize to the cytoplasmic compartment of the cells, but a noted localization of ORF2 into nucleus also occurs implicating regulatory roles in the several aspects of the viral life cycle including, replication, transcription or in virion biogenesis. We are currently further investigating their roles in viral life cycle. P142 Agnoprotein of JC Virus, BK Virus and Simian virus 40 is Released from Cells: Importance of the Dimerization Domain for Release A. Sami Saribas, Jason Saredy, Martyn White, Mahmut Safak (corresponding author: ) Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, USA JC virus (JCV) is the etiological agent of the fatal disease, progressive multifocal leukoencephalopathy (PML), in the central nervous system (CNS), seen in a subset of patients with underlying immunosuppression, including lymphoma and AIDS. JCV specifically infects oligodendrocytes and astrocytes and causes an extensive myelin loss in CNS leading to the neurological manifestations of PML. PML is a rare disease, but the incidence of PML dramatically increased in the era of the AIDS epidemic (5–7 %). PML is also steadily increasing among a group of patients with autoimmune disorders, including multiple sclerosis (MS) and Crohn’s disease, who are treated with immunomodulatory antibodybased therapies. Agnoprotein is an important regulatory protein of several polyomaviruses including JCV, BKV and SV40 and these viruses are unable to replicate efficiently in the absence of this protein. Agnoprotein forms highly stable dimers/oligomers through its Leu/ Ile/Phe-rich alpha-helix domain, which plays a critical role in stability of the protein. In this report, we have investigated whether agnoprotein of BKV and SV40 exhibits similar characteristics with respect to its release from cells as was previously reported for JCV. Our data showed that agnoprotein of BKV and SV40 is also released from cells. We further investigated the regions and amino acid residues responsible for this release by employing mapping and site-directed mutagenesis studies. These studies revealed that amino acid residues Phe31 and Asp38 located within the dimerization domain of JCV agnoprotein play important roles in release. In addition, treatment of cultured cells with a recombinant agnoprotein demonstrated a strong interaction of agnoprotein with the cell surface, suggesting a novel role for this protein during initiation of the next round of the viral infection cycle. This study has been made possible by grants awarded to M. Safak from NIH (1R01NS090949-01A1) and Temple University Drug Discovery Initiative (161398). P143 Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells. Ilker Sariyer, Rahsan Sariyer, Jessica Otte, Jennifer Gordon (corresponding author: ) Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, USA PML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV), which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs). We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF). SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNAbinding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen. Pur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins. Taken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral reactivation leading to development of PML. P144 Serum Inflammatory Markers and Risk of Dementia and Neuropathy among HIV+ adults in Rakai, Uganda Deanna Saylor1, Anupama Kumar1, Gertrude Nakigozi2, Noeline Nakasujja3, Kevin Robertson4, Carlos PardoVillamizar1, Ronald Gray5, Maria Wawer6 (corresponding author: ) 1Department of Neurology, Johns Hopkins University School of Medicine; 2Rakai Health Sciences Program; 3Department of Psychiatry, University of Makarere; 4Department of Neurology, University of North Carolina - Chapel Hill; 5Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health; 6Department of Epidemiology, Johns Hopkins University Bloomberg School of Public Health Background: Systemic inflammation has been linked to risk of HIV-associated neurocognitive disorder (HAND) western HIV+ cohorts. Its relationship to HIV-associated peripheral neuropathy has not been investigated. Objective: We investigated the association between serum interleukin-6 (IL-6) and D-dimer with HAND and neuropathy among HIV+ adults in rural Uganda. Methods: 400 HIV+ antiretroviral therapy naíve adults were included. All participants completed a sociodemographic survey, neurological examination, neurocognitive assessment, and venous blood draw. Neuropathy was defined as the presence of >1 subjective neuropathy symptom and >1 objective sign of neuropathy on examination. Presence of dementia was determined using locally derived normative data and Frascati criteria. Serum D-dimer levels were determined using ELISA, and serum IL-6 levels were determined using singleplex assays. Means of logtransformed IL-6 and D-dimer were compared using t-tests. Quartiles of IL-6 and D-dimer levels were compared using Wilcoxon rank sum tests. Univariate and multivariate logistic regression analyses were used to compare odds of dementia by serum markers. Results: Participants were 53 % male, mean age 35 + 8 years, and mean education 5 + 3 years. Half of participants had CD4 counts <200 and half had CD4 counts 350– 500. Neuropathy was present in 19 % (n = 76), and 16 % (n = 66) had dementia. Participants with CD4 <200 had higher levels of IL-6 (1.77 vs. 1.01; p < 0.001) and a trend toward higher D-dimer levels (13.16 vs. 12.15; p = 0.06). IL-6 was higher among participants with dementia (1.83 vs. 1.32; p = 0.03) but not those with neuropathy. D-dimer did not vary by dementia or neuropathy status. Odds of dementia increased by 39 % for every quartile increase in IL-6 (OR 1.39; 95 % CI [1.04, 1.85]; p = 0.02) after controlling for CD4 count and D-dimer. Conclusions: Systemic inflammation as measured by IL-6 is associated with an increased risk of dementia but not neuropathy in HIV+ adults in rural Uganda. P145 Activation of SLC1A2 promoter using CRISPR/Cas9 gene editing technology Masoud Shekarabi, Prasun Datta (corresponding author: ) Department of Neuroscience, Center for Neurovirology, Lewis Katz School of Medicine at Temple University, Philadelphia, USA Solute carrier family 1, member 2 (SLC1A2) encodes the glutamate transporter 2 (EAAT2) protein primarily expressed in astrocytes that reuptakes excess glutamate from the synaptic cleft to prevent excitotoxicity. EAAT2 plays an essential role in cognitive functions and decreased expression of EAAT2 protein is observed in NeuroAIDS. In the current study, we investigated whether engineered transcriptional activation systems based on CRISPR/Cas9 can be harnessed to activate HIV-1 Tat mediated dysregulation of EAAT2 expression in astrocytes. We have developed a stable astrocytic cell line that expresses the deactivated Cas9 (dCas9) protein which includes dCas9 cassette in frame with the catalytic domain of p300 protein. We designed guide RNAs to target the promoter and induce the expression of EAAT2 protein. Using multiple techniques, we show that the SLC1A2 promoter is induced by the gRNAs in presence dCas9-p300 in the human glioma cell line. Induction of SLC1A2 promoter led to increase in EAAT2 mRNA and protein expression. In addition, we show that co-transfection of gRNAs with dCas9p300 can mitigate HIV-1 Tat induced downregulation of EAAT2 protein expression in primary human fetal brain astrocytes. Collectively, these results demonstrate that CRISPR/Cas9 system can be used for potential induction of EAAT2 expression not only in NeuroAIDS but also in other neurodegenerative diseases such as ALS and Alzheimer’s disease. P146 Epileptogenesis in Patients with Progressive Multifocal Leukoencephalopathy Fabian Sierra Morales 1, Susan Herman2, Mary-Ann Dobrota2, Dhanashri P. Miskin2, Igor J. Koralnik 1 (corresponding author: ) 1Rush University Medical Center; 2Beth Israel Deaconess Medical Center A l t h o u g h u p t o 4 4 % o f p r o g r e s s i v e m u l t i f o c a l leukoencephalopathy (PML) patients will develop seizures, the mechanisms of epileptogenesis in PML remain unclear. We aimed to determine the incidence and risk factors for seizures in PML. In a prospective observational study of PML patients at BIDMC, we reviewed demographics, symptoms, etiology, CSF studies, JCV PCR, brain biopsy results, and CD4 + T-cell counts. We measured JCV-specific T cell response in their blood by intracellular cytokine staining (ICS). Patients underwent neurologic exam, MRI of the brain (including arterial spin labeling and MR spectroscopy) and 256-channel EEG at enrollment and at 3, 6, and 12 months. EEG was acquired on a Geodesics EEG system 400 with 256 channels Hydrocel Geodesic sensor net. EEG findings were co-registered to the 3D rendition of the brain on MRI. Of 29 PML patients, 48 % of patients had only one enrollment visit, and 52 % were followed for 3–12 months. These included 10 % of patients with clinical seizures prior to enrollment and 38 % during the study. EEG showed focal or multifocal slowing in 21 patients (72 %), both focal and diffuse slowing in 7 (24 %), and no abnormalities in 1 (3 %). Focal slowing was concordant with location of PML demyelinating lesions on MRI in 24 patients (83 %). Epileptiform discharges were present on at least one EEG in 13 patients (45 %), most commonly in temporal (10/13, 77 %) and frontal (3/13, 23 %) regions. Epileptiform discharges were concordant with PML lesion location in 12/13 patients (92 %) and were not associated with a history of IRIS. (p = .70). Focal EEG slowing and epileptiform activity was concordant with demyelinating l e s i o n s o f P M L o n M R I i n a l m o s t a l l p a t i e n t s . Demyelinating lesions appear to be associated with cortical irritability and epileptogenesis. Correlation with perfusion MRI, MR spectroscopy and results of the cellular immune response to JCV will be presented. P147 Protective Role of Smoothened Agonist in HIV-associated Neuro-cognitive Disorder. Meera V Singh1, Vir B Singh1, Santhi Gorantla2, Larisa Y Poluektova1, Sanjay B Maggirwar1 (corresponding author: ) 1Department of Microbiology and Immunology, University of Rochester Medical Center; 2Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center Sonic Hedgehog (Shh) signaling is critical during neurogenesis, however; recently, it has also been identified to play an important role in the maintenance of blood brain barrier (BBB) homeostasis in adult CNS. Previous work from our lab suggests that Human Immunodeficiency Virus (HIV) infection causes impaired BBB. Excessive migration of inflammatory/infected leukocytes across the compromised BBB in to the CNS can exacerbate HIV-associated neuro-cognitive disorder (HAND), a chronic neuro-inflammatory condition, which afflicts nearly 50 % of HIV-infected individuals on antiretroviral therapy (ART). In this report, humanized mice were used as a model for HIV infection, to demonstrate the involvement of impaired Shh signaling in HAND. Mice were chronically infected for 10 weeks and were treated with Smoothened Agonist (SAG) for the last week. Results showed reduced expression of Shh and Gli-1 in the brains of infected mice along with astrogliosis, microglial activation, and reduced dendritic arbor. However upon SAG administration, mice showed increased expression of tight-junction proteins (Claudin5, Occludin) and reduced astrogliosis, indicative of better BBB integrity and decreased inflammation. Further the ability of SAG to prevent leukocyte infiltration into the brain during acute infection was assessed. Mice were pre treated with SAG followed by infection with HIV for 2–3 days. Interestingly, SAG significantly reduced the number of leukocytes extravasating into the brain indicating that these mice may eventually have reduced viral burden. Our results emphasize a neuroprotective role for Shh signaling, while revealing SAG as a potential therapeutic agent. These results are also consistent with the outcome of clinical trial (RV254/SEARCH 010), which showed HIV entry into the CNS as early as 8 days of infection. Reduced viremia during this phase, possibly via SAG treatment, might subsequently result in a better prognosis for HAND. P148 Assessing the correlation between the grey and white matter damage and HHV-6 involvement in the frontal and temporal lobes of the brain of the elderly 1Institute of Anatomy and Anthropology, Riga Stradins University; 2Department of Neurosurgery, Stanford University School of Medicine; 3A. Kirchenstein Institute of Microbiology and Virology, Riga Stradins University; 4Department of Pathology, Riga 1st Hospital; 5Latvian State Centre for Forensic Medical Examination Aging is an important risk factor for developing neurodegenerative disorders. High percentage of world’s population is seropositive for human herpesvirus 6 (HHV-6), most commonly the elderly. The immune responses the host can mount against infectious agents undergo age-dependent changes. Microglial cells are the main immune-competent cells of the CNS forming the first line of defense against invading pathogens in case of injury or disease of the brain. Human brain tissue autopsy samples from the frontal and temporal lobes of 25 elderly subjects and 25 controls were u s e d i n t h i s s t u d y. T h e i n c l u s i o n c r i t e r i a w e r e pathomorphological signs of an unspecified encephalopathy on tissue examination. Whole slide scanning, conventional immunohistochemistry using anti-HHV-6 and antiCD68 antibodies and confocal microscopy were applied. Immunostaining intensity was assessed with an additional quantitative estimation of immunopositive cells. Brain tissue samples were assayed for HHV-6 using nested and real-time PCR. SPSS 23.0 program was used for statistical analysis. The HHV-6 DNA was detected in the frontal and temporal lobes respectively of the following number of cases - encephalopathy group - 36 % (9/25), 44 % (11/ 25); and control group - 16 % (4/25), 38.8 % (7/25). In both regions studied, there were significantly higher (p = <0.001) number of HHV-6 positive gray matter astrocytes, oligodendrocytes, microglial cells and neurons in the encephalopathy group when compared to controls. By contrast, there were significantly (p = <0.001) more HHV-6 positive oligodendrocytes and microglial cells found in the white matter when compared to the gray matter. An increased number of activated microglial cells were detected in the white matter of the frontal and temporal lobes of the encephalopathy group. More neuronal and oligodendroglial HHV-6 positivity was detected in the gray and the white matter, respectively, demonstrating heterogeneity of damage to the cortex and subcortical white matter. P149 In vivo visualization and quantification of Spatial-Temporal kinetics of Chimeric HIV infection and the efficacy of Truvada as PrEP Jiasheng Song1, Alexander G. White1, Yonggang Zhang2, Fei Yu1, Wenhui Hu2, Won-Bin Young1 (corresponding author: ) 1Department of Radiology, University of Pittsburgh School of Medicine; 2Department of Neuroscience, Temple University School of Medicine, Philadelphia, PA, USA Visualizing primary sites of HIV-1 infection and viral reservoirs longitudinally would be extremely useful for the development of antiretroviral drugs and vaccines. Currently, surrogate animal models are the major platform for testing newly developed antiretroviral therapy (ART) and vaccines against HIV infection. To establish a lower-cost high-throughput drug screening platform in conjunction with studying how viral reservoirs are formed and maintained, we have employed a chimeric HIV-1 virus which contains a replacement of the HIV gp120 protein with gp80 from the ecotropic murine leukemia virus. This allows for infection of conventional mice without the need of expensive humanized mice. Insertion of the enhanced firefly luciferase gene in this chimeric HIV enables the infected cells to be visualized via in vivo bioluminescence imaging. We systemically imaged the spatialtemporal biodistribution of the infected cells to reveal the viral infection kinetics during the first 24 h and followed the infection for 107 days. The biodistribution of infected cells, mainly neutrophils, was visualized and quantified longitudinally using bioluminescence imaging to illustrate the migration of infected cells in vivo. Ex vivo imaging revealed the infected cells reside in liver, gastrointestinal (GI) tract, spleen, peripheral lymph nodes, and nasal mucosa. The viral RNA in the spleen was linearly correlated to the photon flux output measured from the light emission from the spleen during whole animal imaging. We also showed that this imaging/chimeric HIV platform can be used directly to evaluate the efficacy of Truvada as Pre-Exposure Prophylaxis against HIV infection in living animals. P150 Substance P Mediated Chemokine Production Promotes Migration of Human Monocytes Sergei Spitsin1, Steven D. Douglas2 (corresponding author: ) 1Division of Allergy and Immunology, The Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA; 2Division of Allergy and Immunology, The Children’s Hospital of Philadelphia Research Institute, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA Substance P (SP) is a neuropeptide which plays a role in neurotransmission in the central and peripheral nervous system and has immunomodulatory properties. SP is a member of the tachykinin family of neuropeptides and acts through interaction with neurokinin-1 receptor (NK1R) which is expressed by a variety of cells including lymphocytes and monocyte/ macrophages. SP has physiological and pathophysiological roles in both CNS and peripheral tissues and is involved in cross talk between nervous and immune systems in various conditions including HIV and SIV infection. Increased SP levels were demonstrated in plasma of HIV positive individuals as well as in the CNS of SIV infected non-human primates. SP increases HIV infection in macrophages through interaction with NK1R. The SP effect on immune system is both pro- and anti-inflammatory and includes upregulation of a number of cytokines and cell receptors. We determined whether there is interplay between monocyte exposure to SP and their recruitment into CNS. We demonstrated that exposure of both human monocytes/macrophages and PBMC to SP leads to increased production of chemokines including MCP-1 which is expressed only by cells of myeloid lineage. This effect was inhibited by the NK1R antagonist, aprepitant. Exposure to conditioned media from SP treated PBMC resulted in increased monocyte migration through semipermeable membrane and in vitro human BBB model. Cell migration was blocked by anti-MCP-1 antibodies. Conclusions: The exposure of monocyte/ macrophages to a greater levels of SP during HIV infection may lead to increased MCP-1 production in the CNS and promote further monocyte migration into CNS thus contributing to HIV infection of the brain. The transmigrated monocytes may be a major source of the CNS reservoir in HIV infected individuals and factor contributing to CNS inflammation. Supported by NIH PO1 MH-105303, UO1 MH-090325, R21 AI-108296, P30 MH-097488 to SDD P151 A role for BACE1 in HIV-associated neurotoxicity Anna Stern1, Patrick Gannon1, Benjamin Gelman2, Dennis Kolson1, Kelly Jordan-Sciutto1 (corresponding author: ) 1The University of Pennsylvania; 2University of Texas Medical Branch HIV-associated neurocognitive disorder (HAND) persists in 30–50 % of HIV+ patients despite effective viral suppression by combined antiretroviral therapy. Though a distinct disorder, HAND has symptoms and neuropathological features in common with Alzheimer’s Disease (AD). A hallmark of AD pathology is accumulation of amyloid-beta (Abeta), which is generated by cleavage of amyloid precursor protein (APP) by the beta-secretase BACE1. BACE1 is increased in AD brain, and BACE1 inhibitors reverse neuronal loss and cognitive decline in animal models of AD with human trials underway. Although some studies have found evidence of altered APP processing in HIV+ patients, it is unknown whether increased BACE1 or accumulation of oligomeric Abeta are a feature of HAND. As HIV does not infect neurons, neurotoxicity is attributed to factors released from HIV-infected macrophages including glutamate, and NMDA receptors mediate HIV-associated neurotoxicity in vitro. Herein, we hypothesize that HIV-associated neurotoxicity is mediated by NMDA-dependent elevation of BACE1 and subsequent altered processing of APP. In support of this hypothesis, we have observed elevated levels of BACE1 and Abeta oligomers in CNS of HIV+ patients. As a model of HIV-induced neurotoxicity, we treated primary rat neurons with supernatants from HIV infected monocyte derived macrophages (HIV/MDMs). We observed elevation of BACE1 protein levels in both HIVMDM- and NMDA-treated neurons, and HIVMDMinduced BACE1 increases were blocked by NMDA receptor inhibition. Furthermore, blocking BACE1 activity with a BACE1 inhibitor abrogated HIV/MDM-induced and NMDA-induced neurotoxicity. These findings suggest that increased BACE1 and resultant Abeta production may contribute to HAND neuropathology, and inhibition of BACE1 may have therapeutic potential for HAND patients. P152 Research Training in Neurovirology: Supporting Pathways to Success National Institute of Mental Health, Division of AIDS Research This session represents our annual efforts at the current I S N V m e e t i n g t o s u p p o r t r e s e a r c h t r a i n i n g i n neurovirology. The session is designed to provide new and early stage research investigators with the tools necessary to continue along the path of competitive research support and transition to independence. As in previous meetings, active T32 and other predoctoral and/or postdoctoral trainees will briefly present data and research directions for their current mentored research projects and will be offered feedback for improvements. Preceding and subsequent to the trainee presentations, the symposium content will include: (1) Introductory presentation on neurovirology research trajectories, with special attention to barriers faced by e m e r g i n g y o u n g i n v e s t i g a t o r s t o s u c c e s s i n neurovirology at various levels (individual, institutional, organizational, systemic), and (2) Panel discussion including predoctoral, post-doctoral and faculty investigators on strategies for successfully navigating obstacles and developing potential solutions on the journey to a successful research career. One of the intentions of this roundtable discussion will be to facilitate mentoring relationships at multiple levels. P153 EVALUATION OF COMBINATION LONG-ACTING NANOFORMULATED ANTIRETROVIRAL THERAPY IN EARLY HIV-1 INFECTED huPBL NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ MICE Hang Su, Prasanta Dash, Mariluz Arainga, Benson Edagwa, JoEllyn McMillan, Larisa Poluektova, Santhi Gorantla, Howard Gendelman (corresponding author: ) Early clinical intervention with combination antiretroviral therapy (ART) in human immunodeficiency virus type one (HIV-1) infected people was demonstrated to have sustained benefits by lowering viral set points, protecting CD4+ T lymphocytes and limiting viral reservoir pools that include central nervous system (CNS), which holds limited ART excess, persistent viral replication and cellular activation, leading to continued neuropsychological impairment, causing HIV-associated dementia (HAD). Notably and as shown in our prior studies cell-targeted nanoformulated ART (nanoART) improves pharmacokinetic and pharmacodynamics profiles and reduces local and systemic drug toxicities in experimental models of HIV/ AIDS. In addition, with optimized size, shape and protein and lipid coatings, nanoART can circumvent the blood– brain barrier (BBB) to facilitate CNS-directed drug delivery. These drugs are being considered in a step-wise drug regimen for viral eradication. In the present study, we administrated long-acting nanoformulated combinations of folic acid-decorated cabotegravir, lamivudine and abacavir to human peripheral blood lymphocyte (PBL) reconstituted NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice 24 or 72 h after HIV-1 infection. Replicate mice received free drugs at equivalent doses. Animals were sacrificed 2 weeks after HIV-1ADA infection with blood and tissues harvested for viral measurements. NanoART-treated animals demonstrated 75 % recovery of CD4+ T lymphocytes calculated from a total CD3+ cell pool. Immunohistochemical tests showed HIV-1p24+ cells (~80/1000HLA-DR+ cells) only in untreated infected animals. NanoART suppressed HIV-1gag RNA expression to <10copies as assessed by real-time RT-PCR in brain, spleen, live, lung and kidney in 90 % animals while total viral DNA remained detectable. Limited tissue proviral DNA was seen in nanoART treated animals with significant reduction, up to or exceeding 85 %, in plasma viral RNA levels was observed in 24 h nanoART-treated group (<20 copies/ml). Early treatment of combination nanoART in infected PBL mice can effectively suppress HIV-1 growth in both central and peripheral viral sanctuaries while preventing CD4+ T lymphocyte loss. P154 Clinical and functional characterization of viral single nucleotide polymorphisms (SNPs) within the HIV-1 LTR associate with increased virus persistence in the Drexel Medicine CARES Cohort Neil Sullivan1, Michael Nonnemacher1, Vanessa Pirrone1, Rui Feng2, Brian Moldover3, William Dampier1, Shendra Passic1, Jean Williams1, Benjamas Aiamkitsumrit1, Wen Zhong1, Brandon Blakey1, Sonia Shah1, Zsofia Szep4, Jeffrey Jacobson5, Brian Wigdahl1 (corresponding author: ) 1Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel U n i v e r s i t y C o l l e g e o f M e d i c i n e ; 2 D e p a r t m e n t o f Biostatistics and Epidemiology, Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine; 3B-Tech Consulting, Ltd; 4Division of Infectious Diseases and HIV Medicine, Department of Medicine, Drexel University College of Medicine; 5Department of Medicine, Section of Infectious Disease, Lewis Katz School of Medicine, Temple University The HIV-1 LTR is continuously under selective pressure and LTR SNPs can alter viral transcription in a cell-type dependent manner. To elucidate the clinical and functional impact of HIV-1 SNPs, the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort conducted a prospective, longitudinal study on >500 HIV-1infected patients. Numerous SNPs were strongly correlated with clinical disease parameters, such as CD4+ T-cell count and viral load. Of interest, LTR position 108, a COUP/AP1 binding site, increased in frequency in patients with high viral loads and low CD4+ T-cell counts. Electrophoretic mobility shift assays (EMSAs) functionally demonstrated differential transcription factor (TF)/DNA binding profiles with Jurkat T-cell and monocytic U-937 nuclear extract (NE) when the nucleotide at position 108 is changed. The binding site with an A at position 108 (108A) formed 3 complexes while a G at this position (108G) formed 4 complexes with Jurkat NE. Three complexes were formed with both constructs when U-937 NE were used in the EMSAs. JASPER and supershift EMSAs support the presence of GATA-2, ETS-1, AP-1, and COUP binding to the sequences surrounding 108. Transient expression analyses with an LAI LTR containing a 108G showed increased transcription as compared to 108A in monocytic U-937 cells but not in Jurkat T cells. This observation correlates with the increased viral load and persistence associated with the 108G change in specific HIV-1-infected individuals. These results demonstrate that mutations are occurring in individuals on antiretroviral therapy that are likely clinically and functionally important. P155 IFNbeta PROTECTS NEURONS IN A CCL4-DEPENDENT FASHION AGAINST HIV-1 GP120-INDUCED INJURY Victoria Thaney1, Alan O’Neill1, Melanie Hoefer1, Ricky Maung1, Marcus Kaul1 (corresponding author: ) 1Sanford Burnham Prebys Medical Discovery Institute Human immunodeficiency virus-1 (HIV-1) invades the central nervous system (CNS) soon after peripheral infection and can cause HIV-associated neurocognitive disorders (HAND). Type I interferons are critical mediators of anti-viral immune responses and interferon-beta (IFNbeta) has been implicated in the control of HIV and SIV infection in the brain. However, whether IFNbeta has neuroprotective activity in the context of HIV-mediated neuronal injury is unknown. In the present study, we used HIVgp120 transgenic mice (HIVgp120tg) as a model for HIV-induced brain injury that may underlie the development of HAND. Analysis by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR) detected an IFN response in gp120 transgenic brains with IFNbeta mRNA significantly increased at 1.5 months, but not at 3 and 6 months. Therefore, we examined whether recombinant IFNbeta treatment can prevent neuronal injury and/or glial activation in vivo and in mixed neuronal-glial cerebrocortical cell cultures. In vivo, we administered IFNbeta intranasally in order to maximize IFNbeta delivery to the brain and to minimize side effects in peripheral tissues. We found that a 4-week IFNbeta treatment triggered an IFN response that included increased CCL4 expression. Neurohistological analysis revealed that IFNbeta treatment prevented loss of neuronal dendrites and presynaptic terminals in the frontal cortex and hippocampus in gp120tg brains. Similarly, treatment of mixed neuronalglial cerebrocortical cells with IFNbeta provided complete neuronal protection against recombinant gp120 toxicity. Neuroprotection was dependent on IFNalpha receptor 1 (IFNAR1) and CCL4, as IFNAR1 deficiency and neutralizing antibodies against CCL4, respectively, abolished the neuroprotective effects of recombinant IFNbeta. Taken together, these results identify IFNbeta as a neuroprotective factor that can ameliorate HIV gp120-induced brain injury via induction of CCL4 and be delivered in vivo using i n t r a n a s a l a p p l i c a t i o n . S u p p o r t e d b y N I H R 0 1 MH087332, MH104131 and MH105330 P156 HIV-1 Tat protein activates microglia through defective mitophagy Annadurai Thangaraj, Palsamy Periyasamy, Shilpa Buch (corresponding author: ) D e p a r t m e n t o f P h a r m a c o l o g y a n d E x p e r i m e n t a l Neuroscience, University of Nebraska Medical Center, Omaha, NE, ZIP: 68198 HIV-1 invades CNS early in the course of infection and primes a cascade of neuroinflammatory responses by activating immune cells. Since HIV-1 transactivator protein, Tat has been well-documented to activate glial cells, the objective of this study was to explore whether Tat-mediated activation of microglia also involved mitophagy. Our results demonstrated that exposure of microglia to Tat resulted in cellular activation by altering the mitochondrial membrane potential and by increasing the expression of mitophagy markers, Pink1 and Parkin in both BV-2 cells and mouse primary microglial cells. Increased Pink1 accumulation on the defective mitochondria has been shown to recruit cytosolic Parkin to the site of damaged mitochondria for their clearance via mitophagy. We also showed that exposure of microglia to Tat resulted in increased expression of DRP1, thereby escalating the fission of damaged mitochondria. Tat exposure also upregulated the expression of autophagosome markers, Beclin1 and LC3-II, indicating thereby that increased levels of Pink1, Parkin, and DRP-1 proteins likely tether the damaged mitochondria to the LC3-positive phagophore for mitophagosome formation. These findings were validated by imaging studies which demonstrated increased co-localization of damaged mitochondria with the LC3 puncta. Intriguingly, Tat exposure also increased the expression of p62, signifying thereby a possible blockade of mitophagy flux, that in turn, results in accumulation of mitophagosomes. This phenomenon was further validated by mRFP-EGFP-LC3 transfection wherein the results confirmed Tat-mediated defective mitophagy. Interestingly, Tat-mediated activation of microglia correlated with increased expression of TNF-alpha, IL-1beta, and IL-6. Using both pharmacological and gene-silencing approaches to block either autophagy/mitophagy, our findings demonstrated that Tatinduced activation of microglia involved sequential activation of mitophagy with defective clearance. In summary, Tat activates microglia by both increasing mitochondrial damage and defective mitophagy. Interventions aimed at blocking mitochondrial dynamics could thus provide promising therapeutic targets for abrogating Tatmediated neuroinflammation. (Supported by DA040397; DA035203) P157 Reduced cellular energy as a potential marker of HIV brain latency related neuropathogenesis: a pilot study Michael Tobia1, Simon Jones2, Michael Lovelace2, Caroline Rae1, Lucette Cysique1, Bruce Brew1 (corresponding author: ) 1UNSW Australia; Neuroscience Research Australia; 2UNSW Australia; Applied Neurosciences Program, Peter Duncan Neuroscience Research Unit, St. Vincent’s Centre for Applied Medical Research Background: In virally suppressed HIV-infected (HIV+) the cause of brain injury is not known. One hypothesis is that the brain acts as a reservoir for latent HIV infection, and that the burden of HIV brain latency causes a cascade of neuropathological events. In the current study we explore whether major brain metabolites may serve as markers of brain HIV latency, defined by CSF BCL11b (a microglia cell transcription factor that inhibits HIV transcription). Methods: The study included 26 HIV+ men (mean age 57 years old; stable on cART ≥6 months, nadir CD4+ lymphocyte count ≤350 cells/mm3, HIV infection ≥5 years; 96 % undetectable <50 cp/mL; 100 % <100cp/mL). All undertook lumbar puncture; a 1H Magnetic Resonance Spectroscopy (MRS) scan. CSF samples were analyzed for BCL11b, neopterin, NFL, and tat. All were had undetectable HIVRNA at <50 cp/mL. 1H MRS included measurements of N-acetyl Aspartate (NAA), choline, creatine, Myo-inositol, glutamine/glutamate in the frontal white matter, posterior cingulate cortex, and caudate nucleus area. MRS spectra were measured with reference to the unsuppressed water signal and quantified using JMRUI V.03. Results: In a series of adjusted (for neopterin, NFL and tat) regression models; we found that a higher CSF BCL11b was consistently associated with lower frontal white matter creatine (adjusted for neopterin, Std beta = −.30; p = .15; adjusted for NFL, Std beta = −.51; p = .03; adjusted for tat, Std beta = −.47; p = .02). Conclusions: Frontal white matter reduced cellular energy may indicate ongoing HIV brain latency related neuropathogenesis. These pilot data need to be further tested in a larger sample. P158 Neutralization of interferon-alpha by B18R in a mouse model of HIV associated neurocognitive disorders 1Emory University School of Medicine and Atlanta VA Medical Center; 2Atlanta VA Medical Center; 3Arizona State University; 4Emory University; 5Emory University and the Atlanta VA Medical Center; 6Meiogen Infection with HIV commonly causes HIV associated n e u r o c o g n i t i v e d i s o r d e r s ( H A N D ) d e s p i t e l i f e prolonging combined antiretroviral therapy (cART). There is substantial evidence that interferon-alpha plays a role in cognitive impairment in HAND. We have previously shown that a decoy protein that sequesters type I interferons, B18R, ameliorates HIV induced histopathology. We tested if co-administration of B18R with cART provides enhanced protection from HIV encephalitis in comparison to B18R or cART treatments alone in a mouse model of HAND. After 10 days of treatment mice were sacrificed and brains extracted and frozen for tissue sectioning. The data suggests that B18R simultaneously administered with cART reduced encephalitis markers more than either of the two treatments administered alone. In a separate set of experiments, B18R was tested for its ability to reverse behavioral abnormalities of HAND mice using an Object Recognition Test (ORT). The ORT was administered to HAND mice and controls before and after treatment with B18R. Prior to B18R treatment HAND mice were behaviorally impaired on the ORT. Then HAND mice received either B18R or saline subcutaneously twice a day for 5 days prior to repeat ORT. Performance was significantly improved in B18R treated HAND mice compared to saline treated mice. Pathological studies in these ORT, B18R treated and untreated HAND mouse groups are ongoing. Because of its ability to act in concert with cART as well as reverse behavioral abnormalities in HAND mice, B18R is a potential therapeutic agent for a Phase I trial in HAND patients. P159 The epidermal growth factor counteracts the effects of HIVtat on the expression of endogenous retroviruses of the W family in human astrocytes Elena Uleri, Gabriele Ibba, Claudia Piu, Maurizio Caocci, Caterina Serra, Antonina Dolei (corresponding author: ) Department of Biomedical Sciences, University of Sassari The pathogenic mechanisms of HIV-related neurodegeneration are not fully elucidated, and include both host and viral mechanisms. The epidermal growth factor (EGF) has been proposed to control the phenotypic features of adult astrocytes. Activation of EGFR, the EGF receptor, was proposed as a master pathway of astrocyte activation in response to different neural injuries. We showed previously that HIVtat activates the neuropathogenic and immunopathogenic MSRV and Syncytin-1 elements of the HERV-W family of endogenous retroviruses, in human macrophages and in astrocytes. We hypothesized that these elements could contribute to the pathogenic phenomena leading to HIV-related neurodegeneration. The mechanism of HERV-Ws stimulation by Tat is an indirect one, through interaction with Toll-like Receptor-4 (TLR4), without internalization, and induction of TNFalpha. It was shown that HIVtat interacts with the 1–6 EGF-like repeats of Notch proteins, with a possible link with AIDS pathogenesis. Therefore, we exposed the PHFA, SVG and U87MG human astrocyte cells to HIVtat, EGF and other stimuli, and the expression of MSRV and Syncytin-1 HERV-W transcripts was evaluated by real time RT-PCR. The results indicate that EGF counteracts the effects of HIVtat on the HERV-Ws, by interfering with the induction of TNF-alpha by HIVtat. The EGF-mediated counteraction of HIVtat effects is prevented by pre-exposure to antibody against the EGF-R. P160 JC polyomavirus expression and bell-shaped regulation of its SF2/ASF suppressor in multiple sclerosis patients under therapy with Natalizumab Elena Uleri, Gabriele Ibba, Claudia Piu, Maurizio Caocci, Caterina Serra, Antonina Dolei (corresponding author: ) Department of Biomedical Sciences, University of Sassari Natalizumab is effective against multiple sclerosis (MS), but r a r e l y a s s o c i a t e d w i t h p r o g r e s s i v e m u l t i f o c a l leukoencephalopathy (PML), fatal brain disease due to the JCV polyomavirus. The SF2/ASF (splicing factor2/ alternative splicing factor) inhibits JCV in glial cells. We wondered about SF2/ASF modulation in the blood of Natalizumab-treated patients, and if this could influence JCV reactivation. A 2-year study was performed on blood cells from Natalizumab-treated and Fingolimodtreated MS patients. A bell-shaped SF2/ASF regulation was observed only under Natalizumab: SF2/ASF was upregulated, during the first year, only in JCV DNApositive patients, or with high anti-JCV antibody, with enrichment of SF2/ASF-negative B-cells. The expression of the JCV T-Ag protein in circulating B cells was inversely related to SF2/ASF protein expression. The increase of SF2/ASF-negative B-cells, parallel to JCV activation, during the second year of therapy with Natalizumab, but not with Fingolimod, may help explaining the increased risk of PML with Natalizumab, and not with Fingolimod. P161 JAM-A and ALCAM Mediate Preferential Transmigration Across the BBB of HIV-infected HIV + CD14+CD16+ Monocytes: Continued Viral Seeding of the CNS and HAND. 1Department of Pathology, Albert Einstein College of Medicine; 2John A Burns School of Medicine, University of Hawaii at Manoa; 3De pa rtme nt of Mo lec ul ar an d Comparative Pathobiology, Johns Hopkins University; 4Department of Medicine, Albert Einstein College of Medicine; 5Department of Neurology, Mount Sinai School of Medicine HIV associated neurocognitive disorders (HAND) affect >50 % of HIV-infected individuals. HAND is mediated by entry of infected CD14+CD16+ monocytes into the CNS that establish and reseed CNS viral reservoirs, even when people have undetectable viral loads. CD14+CD16+ monocytes transmigrate across the blood–brain barrier (BBB) into the CNS in response to chemokines, including CCL2. Viral reservoirs release early viral proteins and cytokines, even during active cART, that mediate persistent neuroinflammation and neuronal damage leading to HIV neuropathogenesis. To develop therapeutics that can prevent viral (re)seeding of the CNS and subsequent HAND, we examined the mechanism of entry of these HIV-infected cells into the CNS. We show for the first time that HIV-infected HIV+CD14+CD16+ monocytes preferentially transmigrate across the BBB in comparison to uninfected but HIV-exposed HIVexpCD14+ CD16+ monocytes, both by p24 FACS and HIV DNA. We also show that this is mediated, in part, due to increased junctional proteins JAM-A and ALCAM on HIV+CD14+ CD16+ monocytes. We also showed that JAM-A and ALCAM blocking antibodies inhibit transmigration across the BBB of HIV+CD14+CD16+ monocytes. In addition, CCR2, the only known CCL2 receptor on monocytes, is increased on CD14+CD16+ monocytes from HIV-infected individuals with HAND, but not on those with impairment due to other causes. Using neuroimaging, we showed that increased CCR2 on CD14+CD16+ monocytes correlates with decreased neuronal activity in the basal ganglia. Additionally, using HIV DNA analysis we found that increased CCR2 correlates with increased peripheral PBMC HIV DNA. This provides strong evidence that CCR2 identifies individuals with cognitive impairment due to HIV, and that CCR2 may be a HAND biomarker. To examine CCR2 in addition to JAM-A and ALCAM as a therapeutic target, we will use Cenicriviroc to prevent transmigration of HIV+CD14+CD16+ monocytes into the CNS. It is hoped that these strategies will result in decreased neuronal damage and inflammation, and, HAND. P162 Characterization of Zika virus infection in Human Astrocytes Courtney Veilleux, Eliseo Eugenin (corresponding author: ) Public Health Research Institute (PHRI), Rutgers the State University of New Jersey, Newark, NJ, USA; Department of Microbiology, Biochemistry, and Molecular Genetics, Rutgers the State University of New Jersey, Newark, NJ, USA Zika viral infection is correlated with an increased rate of microcephaly in children born of mothers infected during pregnancy and neurocognitive disorders in adults. Although documented as a mild febrile illness in less than two dozen cases prior to 2003, the Brazilian Zika outbreak strain spread to over 40 counties and territories since its suspected origin in Brazil in 2015 and has infected over 7300 in the US and US territories since June 2015. Additionally, 479 pregnancies within the continental US and 493 pregnancies in US territories have had evidence of Zika infection during pregnancy. Microcephaly as a result of Zika has been identified in more than a dozen US births. The pathogenesis of Zika and its effects on the developing brain are unknown. Previous research in our laboratory has demonstrated that astrocytes play a significant role in perpetuating viral infection within the brain. Our laboratory has identified astrocytes as key viral reservoirs and mediators of apoptosis in CNS cells during viral infection. Thus, our objective is to characterize whether Zika infection in human fetal astrocytes alters survival or glial and neuronal cells. We hypothesize that Zika infection of human fetal astrocytes causes apoptosis that contributes to the devastating CNS consequences of this virus. Our results indicate that Zika induces apoptosis of glial and neuronal cells. Altogether, these results illustrate a distinct mechanism of Zika infection in human fetal astrocytes. Understanding infection in primary CNS resident cells is critical in understanding the pathogenesis of Zika infection in the developing brain. P163 Modulation of Tryptophan degradation via the kynurenine pathway during HIV and SIV infection 1Department of Neuroscience, Lewis Katz School of Medicine Temple University; 2Bioqual Tryptophan (TRP) is an essential amino acid and precursor for the de novo synthesis of nicotinamide adenine dinucleotide (NAD), as well as serotonin and melatonin, by two different pathways. TRP is catabolized by the kynurenine (KYN) pathway to produce NAD and the methoxyindole pathway to generate serotonin and melatonin. Increased KYN/TRP ratios have been associated with the progression of acquired immune deficiency syndrome (AIDS) as determined by association with lower CD4/CD8 ratios, reduced T cell recovery after initiation or cART, and increased mortality risk. Activation of the KYN pathway as well as the neurotoxic metabolite quinolinic acid has been implicated in the pathogenesis of HIV associated neurocognitive disorders (HAND). We identified an increase in the KYN/TRP ratios in the SIV macaque model with a positive correlation with viral load and soluble CD163 in plasma, with the latter suggesting the role of macrophage activation in this process. In an effort to modulate the K/T pathway, and decreased tryptophan catabolism, we performed a dose escalation study with the NAD salvage pathway precursor, nicotinamde riboside (NR), in SIV infected macaques. NR treatment significantly reduced the mean fluorescence intensity and percent frequency of CD14+/CD16+ monocytes as determined by flow cytometry, in both SIV infected and uninfected animals. This monocyte subset has been implicated in the pathogenesis of HIV infection, HAND, as well as other HIV associated comorbidities. In uninfected animals NR treatment had no significant impact on the KTR relative to pretreatment values. In infected animals, however, NR treatment appeared to increase KTR at 100 mg/kg dose, and decrease CD4/CD8 ratio at the highest dose (400 mg/kg). The regulation of the kynurenine pathway appears to be complex, yet an attractive target for HIV therapeutics. Understanding the mechanism by which HIV modulates the KYN/TRP pathway could provide new therapeutic targets for the treatment of HIV and HAND. P164 A novel murine model of enterovirus-71 (EV-A71) -induced neurogenic pulmonary edema Carla Bianca Luena Victorio1, Yishi Xu1, Qimei Ng1, Beng Hooi Chua2, Sylvie Alonso3, Vincent Chow3, Kaw Bing Chua1 (corresponding author: ) 1Temasek Lifesciences Laboratory; 2Curtin University; 3Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore Enterovirus 71 (EV-A71) is now the most common neurotrophic enterovirus following the near eradication of polioviruses. It causes Hand, Foot, and Mouth Disease (HFMD) - a self-limiting febrile illness - and occasional fatal neurological disease in children. Current animal models of EV-A71 infection are not clinically authentic. They fail to replicate the full spectrum of human diseaseäóîspecifically that of neurogenic pulmonary edema (NPE), the major cause of EV-A71 infection-related child mortalityäóîand lack key pathological features observed in fatal human cases. In order to more fully understand the neuro-pathogenesis of severe EV-A71 infection, especially with respect to NPE, our group generated novel mouse-cell-adapted (MCA-EV-A71) viral strains that productively infected both primate and rodent cell lines (Victorio et al., 2014) and utilize the physiologically expressed viral receptor expressed in rodents (mSCARB2) to infect murine cells (Victorio et al., 2016a). We subsequently used these strains to infect 1-week-old BALB/c mice. The infected mice not only exhibited severe, acute lethal disease, but also induced a spectrum of neurological deficits, including that of NPE. We noted signs of respiratory distress and upon necropsy, gross pathological observations confirmed pulmonary edema. Pathological findings in the lung tissues include focal haemorrhagic lesions and accumulation of proteinaceous fluid in the alveoli; while CNS tissue sections revealed massive neuronal destruction and accumulation of viral antigens in the brainstemäóîmost notably in the medulla oblongata (Victorio et al., 2016b). We also observed high levels of serum catecholamines, confirming NPE. Thus, we have developed an authentic mouse model of EV-A71 infection that recapitulated the wide spectrum of clinical signs observed in patients as well as the underlying pathological features observed in fatal cases (Wong et al., 2008). This model could aid in the further understanding of the pathogenesis of EV71-induced neurological diseases and in the development of nov el therapies and prophylactics. P165 Detection of HIV-1 DNA in brain tissue of virally suppressed patients supports the existence of a persistent CNS viral reservoir Emma L. Wanicek1, Lachlan R. Gray2, Wan-Jung Cheng1, Jacob D. Estes3, Sharon R. Lewin4, Paul R. Gorry5, Melissa J. Churchill6 (corresponding author: ) 1Centre for Biomedical Research, Burnet Institute, Melbourne, VIC; 2Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 2. Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, VIC; 3AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, MD; 4Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, VIC 2. The Peter Doherty Institute for Infection and Immunity, The University of Melbourne and Royal Melbourne Hospital, Melbourne, VIC; 5Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 2. Department of Infectious Diseases, Alfred Hospital and Monash University, Melbourne, VIC 3. School of Health and Biomedical Sciences, RMIT University, Melbourne, VIC 4. Department o; 6Centre for Biomedical Research, Burnet Institute, Melbourne, VIC 2. Department of Medicine, Monash University, Melbourne, VIC 3. Department of Microbiology, Monash University, Clayton, VIC Combination antiretroviral therapy (cART) has transformed patient care and disease outcomes for millions of HIV-1 patients globally. However, cART alone is unable to eradicate HIV-1 due to the persistence of numerous viral reservoirs throughout the body. The central nervous system (CNS) is a potential viral reservoir but is difficult to study due to limitations in access to brain tissue. Here, we have applied a novel and highly sensitive (single copy sensitivity) in situ hybridization technique called DNAScope, to detect and visualize HIV-1 DNA in the brain tissue from HIV-infected individuals on suppressive cART. Using a comprehensive bank of tissue samples from clinically well-defined HIV+ patients (n = 45), including both symptomatic and asymptomatic individuals, and representing a range of CNS disease severities, HIV-1 infection was measured by DNAScope. We observed an increase in HIV-1 staining that correlated with increased disease severity. Significantly, HIV-1 DNA was detected in both macrophages/microglia (CD68) and astrocytes (GFAP) in virally suppressed patients. Further, HIV-1 Envelope V3 sequences isolated by laser capture microdissection from macrophages/microglia of 1 patient, demonstrated compartmentalization relative to V3 sequences isolated from PBMCs from the same patient. DNAScope coupled with laser capture micro-dissection, is a powerful technique that allows for the detection, quantification and characterisation of HIV-1 infected cells within the CNS and allows for the characterization of the CNS viral reservoir. This study provides strong evidence that HIV-1 DNA can persist within the CNS of virally suppressed patients and supports the existence of a CNS viral reservoir with important implications for cure research and the development of cure strategies. P166 Investigating the role of Tetherin-dependent pro-inflammatory microparticle release Emily Weber, Meera Singh, Vir Singh, Joe Jackson, Nicole Stirpe, Sanjay Maggirwar ( c o r r e s p o n d i n g a u t h o r : E m i l y A _ We b e r @ U R M C . Rochester.edu) Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry Despite the emergence of combined antiretroviral therapy (cART) that is able to suppress the human immunodeficiency virus (HIV) to undetectable levels in plasma, HIV-positive individuals still face an array of co-morbidities, including an increased risk of neurocognitive disorders. Studies have suggested that chronic inflammation resulting from HIV infection may be due to cytokines/chemokines and viral proteins secreted by activated/infected cells as well as cART administration. In particular, small vesicles known as microparticles (MPs) secreted by various activated cell types, such as monocytes, often contain pro-inflammatory signals, which can contribute to neurodegenerative diseases. Increased inflammatory MP levels have been reported in cART treated HIV-infected individuals. Recent data from our lab suggests that the host restriction factor Tetherin (BST-2/CD317) can sequester and inhibit the release of MPs from monocytes. Further, monocytes from HIV-infected individuals have reduced levels of Tetherin, and plasma samples from these individuals show increased levels of monocyte-derived MPs. Exposure to the HIV viral protein, Tat, can lead to the down regulation of surface levels of Tetherin on monocytes and an increased release of monocyte-derived MPs from healthy individuals. Further, MP sequestration is observed on the surface of monocytes that over express a degradation-resistant mutant of Tetherin, in which the sequence that is used for Vpu-mediated ubiquation is mutated. Additional data from mice harboring a degradation-resistant form of Tetherin shows decreased levels of MPs and increased blood brain barrier function. Information gleaned from these studies will reveal the underlying mechanism of Tetherin-mediated MP release and its role in neurological disease along with other co-morbidities evident in HIVinfected individuals. Furthermore, this work is important in enhancing our understanding of how this novel molecular mechanism of Tetherin-dependent MP release relates to HIV infected individuals, as well as other MP associated diseases. P167 Effects of flaxseed lignin, Secoisolariciresinol diglucose, on endogenous antioxidant pathway in HIV-infected macrophages University of Pennsylvania Macrophages and microglia play pivotal roles in pathogenesis HIV associated neurocognitive disorders. During acute infection, activated and/or infected monocytes infiltrate the central nervous system (CNS) and differentiate into perivascular macrophages. HIV can then spread to microglia, creating an active, protected reservoir in the CNS. The ensuing inflammatory macrophage/microglia activation and secretion of a myriad of toxic factors cause damage to neurons, which are not infected. Thus, current work strives to develop strategies to suppress M/M-driven inflammation and oxidative stress. Numerous studies utilizing exogenous antiinflammatory and antioxidants to mitigate disease progression have been unsuccessful; however, targeting endogenous antioxidant pathways may be more advantageous. A cellular mechanism that mitigates oxidative stress and inflammation is the endogenous antioxidant response (EAR) pathway, which upregulates key antioxidant enzymes, including heme oxygenase 1 (HO-1), to reduce free radicals and suppress tissue damage. In this study, we investigated the role of a flaxseed lignin, Secoisolariciresinol Diglucose (SDG), on viral replication and oxidative stress in HIV-infected macrophages. To evaluate EAR, human monocyte derived macrophages were infected with HIV. By immunoblot, HIV suppressed HO-1 levels, in macrophages, while concurrent treatment with SDG increased HO-1 levels. This was paralleled by increased translocation of nrf2 protein from the cytoplasm to the nucleus and decreased nuclear expre ssion of HO-1’s n eg ative reg ulato r, Ba ch1 . Prolonged HO-1 deficiency in macrophages has been negatively correlated with increased HIV replication in human macrophages. HO-1 levels were decreased in macrophages 12-day post infection. To determine if SDG can reverse prolonged HO-1 deficits, macrophages were pretreated with SDG for 1 h prior to infection and replenished every 3 days. SDG treatment increased HO1 protein levels after 12 days post infection. Given these data, SDG increases endogenous antioxidants pathways and may be a possible adjunctive therapeutic for HIV associated neurocognitive disorders. P168 Insulin Receptor Secretion is not associated with abnormal glucose metabolism in HIV-seropositive women Valerie Wojna1, Miriam Matos2, Rosa J. Rodriguez-Benitez2, Raissa Menendez-Delmestre2, Avindra Nath3, Yamil Gerena4 (corresponding author: ) 1Neurology Division, NeuroAIDS Program, University of Puerto Rico, Medical Sciences Campus; 2NeuroAIDS Program, University of Puerto Rico, Medical Sciences Campus; 3Section of Infections of the Nervous System, NINDS, NIH; 4Department of Pharmacology, NeuroAIDS Program, University of Puerto Rico, Medical Sciences Campus Background: Previously we found that high levels of soluble insulin receptor (sIR) in the plasma of HIV-seropositive women were associated with HIV-associated neurocogntivie disorders (HAND). Although we found evidence of cellular insulin resistance it is not clear if sIR is associated with peripheral insulin resistance as determined by the homeostasis model assessment-estimated insulin resistance (HOMA-IR) index. In this study we investigated if sIR is associated with HOMA-IR. Methods: 50 HIV-seropositive women and 18 controls without history of diabetes were tested for oral glucose tolerance test (including fasting blood sugar [FBS] and insulin levels), glycosylated hemoglobin (HgA1c), HOMAIR, and plasma sIR full-length. Participants were also evaluated for metabolic syndrome with blood pressure, anthropometric measures, and lipid profile. HAND was determined following the HAND nosology criteria. Results: No significant differences were observed between controls and HIV-seropositive women stratified by HAND (normal cognition [18], asymptomatic neurocognitive impairment [ANI,13], and symptomatic neurocognitive impairment [SNI, 19]) regarding age, BMI, HgA1c, insulin and FBS, and HOMA-IR. An association was observed between plasma sIR levels and HAND (p = 0.003), where women with ANI presented with higher levels. No Spearman’s correlation were observed between plasma sIR levels and age, BMI, HIV RNA copies/mL, CD4 cell count, use of protease inhibitors, HGA1c, FBS, insulin levels, and HOMA-IR. Conclusion: Plasma sIR is not associated with standard measures of peripheral insulin resistance such as HOMA-IR. It is possible that the presence of plasma sIR levels and its association with HAND is related to a chronic subclinical cellular insulin resistance. Supported by R21MH095524, R01NS099036, U54MD007587, G12MD007600 P169 In vivo excision of HIV provirus by SaCas9 and multiplex sgRNAs in pre-clinical animal models Chaoran Yin1, Ting Zhang1, Xiying Qu1, Yonggang Zhang1, Raj Putatunda1, Fang Li1, Huaqing Zhao1, Shen Dai1, Xuebin Qin1, Xianming Mo2, Jennifer Gordon1, Won-bin Young3, Kamel Khalili1, Wenhui Hu1 (corresponding author: ) 1Department of Neuroscience, Center for Neurovirology and The Comprehensive NeuroAIDS Center, Temple University Lewis Katz School of Medicine; 2Laboratory of Stem Cell Biology, State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University; 3Department of Radiology, University of Pittsburgh School of Medicine, Pittsburgh Cas9/gRNA-mediated genome editing provides a promising cure for HIV-1/AIDS by excising the proviral DNA from host genome. Here, we demonstrate the feasibility of excising the HIV-1 proviral genome in experimental animal models using saCas9 along with 2–4 multiplex sgRNAs in a single AAV vector. Both duplex and quadruplex sgRNAs/saCas9 can be efficiently packaged into AAV-DJ or AAV-DJ/8 with high titer at 10e13-14 genome copy (GC)/ml. The quadruplex sgRNA/ saCas9 vector outperformed the duplex one in cleaving the integrated HIV-1 genome in cultured neural stem/progenitor cells and various organs of HIV Tg26 transgenic mice. Excision of HIV-1 proviral DNA by quadruplex AAV-DJ/8 (i.v.) was demonstrated by PCR genotyping in liver, lung, brain and spleen of the mice inoculated (i.v.) with chimeric EcoHIV carrying a firefly luciferase reporter (eLuc). Live bioluminescence imaging showed a significant reduction of EcoHIV infection in the neck lymph nodes and whole body. To demonstrate if Cas9/sgRNAs excise HIV provirus in latently infected cells in vivo, we administrated quadruplex sgRNA/saCas9 AAV-DJ/8 (2x10e12 GC/mouse, i.v.) in humanized Bone marrow-Liver-Thymus (BLT) mice with intravaginal inoculation of HIV-1NL-BaL-eLuc. These HIV-infected BLT mice showed significant vaginal infection during the first 3 weeks of inoculation but no observed systemic HIV dissemination for at least 60 days prior to AAV/saCas9 treatment. Successful excision of HIV-1 provirus was detected by PCR genotyping in spleen, lung, heart, colon and brain as well as human thymic organoid on the left kidney as early as 2 weeks after AAV injection. In conclusion, in vivo excision of HIV proviral DNA in solid tissues/organs can be achieved via a single intravenous injection of AAV-DJ/8 carrying quadruplex sgRNA/ saCas9. This is an important step moving forward to the clinical application of the CRISPR/Cas9 gene editing strategy. Supported by NIH grants to KK/WH (R01NS087971), KK (P30MH092177) and WY (R21MH100949). P170 Evidence that Dysfunctional Endolysosomal Biogenesis is Associated with Cognitive Impairment in HIV-Infected Individuals; Agonists of TRPML1 May Restore Lysosomal Function Through Luminal Acidification Seung-Wan Yoo1, Joshua Lisinicchia2, Jacqueline Lovett1, Benjamin B Gelman2, Norman J Haughey3 (corresponding author: ) 1Department of Neurology, Richard T Johnson Division of Neuroimmunology and Neurological Infections, Johns Hopkins University School of Medicine, Baltimore, Maryland; 2Department of Pathology, Neuroscience and Cell Biology, University of Texas Medical Branch Galveston, Texas; 3Department of Neurology, Richard T Johnson Division of Neuroimmunology and Neurological Infections, Johns Hopkins University School of Medicine, Baltimore, Maryland Dysfunction of endolysosomal systems in HIV-Associated Neurocognitive Disorders (HAND) has been implicated by disturbances in transluminal ion gradients, and intraluminal deposition of proteins and lipids. Here, we determined if the Coordinated Lysosomal Expression And Regulation (CLEAR) gene network is impaired by HIV infection using tissues from the National NeuroAIDS Tissue Consortium (NNTC; mini gauntlet sample set). Frontal neocortices were obtained from HIV+ cognitively normal (CN; n = 41), subsyndromic (SS; n = 9), Minor Cognitive Motor Disorder (MCMD; n = 36), HIV Associated Dementia (HAD; n = 29), and HIV- controls (n = 68). All HIV infected subjects showed alterations in the expression of multiple genes coding for hydrolytic enzymes and luminal acidification compared to HIV-. A master regulator of lysosomal biogenesis, transcription factor EB, was increased, and genes related to luminal acidification (chloride channel 7 and transient receptor potential mucolipin1; TRPML1) were decreased in MCMD and HAD compared to CN. These data suggest that HIV infection is associated with a dysregulation of the CLEAR network in neocortex, and cognitive impairment is specifically associated with the induction of lysosomal biogenesis and a reduced ability for luminal acidification. Based on these human data we next used a rodent model of HIV-associated endolysosomal dysfunction (gp120/APP/ PS1 mice) in which the CLEAR network is impaired and lysosomes are engorged with Abeta and sphingomyelin to determine if luminal acidification could rescue lysosomal function. A 26 day intraventricular infusion of the TRPML1 agonist MLSA1 by mini osmotic pump reduced cortical Abeta, sphingomyelin, and restored CLEAR network gene expression to Wt levels. These findings suggest that therapeutic approaches to enhance lysosomal function may protect the CNS in the setting of HIV infection. P171 Drug Use and Age Effects on White Matter Microstructure and Behavior in Treated HIV Infection Thomas Zeffiro1, Erin O’Connor2, Melissa Murray3, Lawrence Kingsley3, James Becker3 (corresponding author: ) 1Temple University; 2Lewis Katz School of Medicine at Temple University; 3University of Pittsburgh Purpose: Although diffusion tensor imaging (DTI) studies have documented white matter (WM) microarchitecture changes following HIV infection, WM properties are also known to change with age and drug use. As the HIV infected population is steadily aging, the effects of age and past drug use may confound attempts to identify specific effects of serostatus on WM microstructure. In a cross-sectional study, we examined the effects of serostatus, age and past drug use on WM microstructure. Methods: Participants included 15 seropositive and 20 seronegative men, ages 54–77, with 12 reporting past drug use. Seropositive participants were all treated with anti-retroviral therapies. Mixed effects models were used to explore effects of serostatus, race, age, and drug use on executive function, learning/memory, attention/ working memory, verbal fluency, processing speed, and motor performance domains. WM microstructure measures included fractional anisotropy (FA) and apparent diffusion coefficient (ADC). Results: Seropositive participants exhibited bihemispheric increases in ADC (p < 0.05 FWE-corrected) and FA (p < 0.05 FWEcorrected). Independent effects of age and past drug use on FA and ADC were seen (p < 0.05 FWEcorrected). Processing speed decreased with age (p < 0.01). Motor performance decreased with age, showing effects of race and an interaction between serostatus and age (all p < 0.01). Executive function, verbal fluency, attention/working memory, and learning/ memory did not show effects of serostatus, age, race or drug use. Conclusions: In a sample of cognitively intact older participants, we observed separate effects of serostatus, age and past drug use on two measures of WM microstructure and associated slowing of movement execution. The relatively wide variation in published studies of HIV infection effects on white-matter microstructure might reflect differences among experimental samples related to age or past drug use. Moreover, WM microstructural changes following HIV infection, while associated with slow motor performance, are not invariably associated with cognitive changes assessed across multiple domains. Author Index Abrams, Rachel, P1 Abimiku, Alash"le, P69 Achim L., Cristian, P25 Achim, Cristian, P18, P42 Agsalda-Garcia, Melissa, P120, P129 Ahooye, Taha, P140 Aiamkitsumrit, Benjamas, P154, P7, P30, P31, P138, P125, P109 Akay Espinoza, Cagla, P2 Akinwumi, Michael, P133 Akkina, Ramesh, P47 Al-Harthi, Lena, P93, P117 Albe, Joseph, P3 Alldred, J., Melissa, P46 Allen, Alex, P40 Allen, Alexander, P4 Allen, Isabel, P111 Alonso, Sylvie, P164 Alt, Jesse, P118 Amancha, Praveen Kumar, P52 Amini, Shohreh, P38, P140 Amor, Sandra, P124 Anastos, Kathryn, P161 Anderson, Monique, P5 Anderson, Brian, P101 Andrews, Allison, P137 Andríçs E., Ibolya, P6 Ansari, Aftab, P33 Antell, Gregory, P7, P31, P125, P109 Arainga, Mariluz, P153 Arancio, Ottavio, P76 Asahchop, Eugene, P8, P133, P97 Atkins, Andrew, P9 Bella, Ramona, P72 Bella, Ramona, P73 Bellizzi, Anna, P13, P14, P15 Bendayan, Reina, P121 Berman, Joan, P161 Berman, W., Joan, P65 Bertrand, Luc, P6 Bevins, Rick, P123 Beyaert, Rudi, P82 BHARGAVAN, BIJU, P16 Bianchet, Mario, P1, P127 Billioux, Bridgette, P86 Bimonte-Nelson, Heather, P158 Birmingham, Amanda, P77 Blackmon, Anna, P68 Blakey, Brandon, P154 Blattner, William, P69 Booze, Rosemarie, P108 Booze, M., Rosemarie, P34, P72, P94, P135 Borjabad, Alejandra, P76, P17 Bouaziz, Serge, P26 Boukli, Nawal, P92 Branton, William, P8, P97 Brew, Bruce, P50, P157 Bryant, Alex, P18 Buch, Shilpa, P19, P156, P123 Budzinski, Allison, P55 Burdo, Tricia, P18, P69 Burke, Deirdre, P115 Byrareddy, Siddappa, P33, P52 Byrd, Desiree, P161 Chauhan, Priyanka, P23 Chauhan, Priyanka, P128 Chen, Xuesong, P60 Chen, Yilan, P71, P73 Chen, Xuesong, P79, P80 Cheng, Wan-Jung, P165 Cheng, Yu, P112 Cherner, Mariana, P69, P74 Cheung, Joseph, P54 Chew, M., Glen, P47 Chiu, Y., Charles, P59 Chow, Vincent, P164, Chow, Dominic, P105 Chua, Beng Hooi, P164 Chua, Kaw Bing, P164 Chung, Cheng-Han (James), P24 Churchill, J., Melissa, P165 Ciavatta, Vincent, P158 Cibrowski, Pawal, P56 Cicalese, Stephanie, P29, P140 Clarke, Penny, P59 Clements, Janice, P132 Clotet, Bonaventura, P114, P113 Coban, Hamza, P25, P42 Cohen, Eric, P97 Colí_n-Cruz, Luis, P45 Colon, Krystal, P134 Coric, Pascale, P26 Cotto, Bianca, P27, P28 Craigie, Michael, P28, P35 Cruz, Michael, P31 Cubano, Luis, P92 Cysique, Lucette, P50, P157 Deleage, Claire, P33 Delgado-Nieves, Andrea, P45 DelValle, Luis, P85 DeMarino, Catherine, P12 Denton, D., Melissa, P99 DePalma, Steve, P21 DePaula-Silva, Ana Beatriz, P37 Dermody, Nadene, P50 Deshmane, Satish, P27, P38 Desimone, Matthew, P4 Desplats, Paula, P25, P42 Devlin, Kathryn, P31 Dickens, Alex, P56, P81 Diez-Quevedo, Crisanto, P113 Dimitriadis, Emilios, P127 Dobrota, Mary-Ann, P146 Doh, Roland, P74 Dolei, Antonina, P160, P159 Donadoni, Martina, P29 Dorskind, Joelle, P32 Doty, Daniel, P37 Doucet-O'Hare, Tara, P39 Dougier, Hei-Lanne , P22 Douglas, D., Steven, P150 Dulcis, Davide, P77 Dutta, Ranjan, P124 Earl, Joshua, P9, P30 Edagwa, Benson, P49, P153 Egan, Kevin, P40 Ehrlich, Garth, P31 El-Hage, Nazira, P12 El-Hage, Nazira, P138 El-Kamary, Samer, P69 Ellis, Ronald, P55, P18 Ellis, J., Ronald, P63 Elrod, John, P27 Endsley, Janice, P44 Engle, Elizabeth, P98 Enose-Akahata, Yoshimi, P5 Estes, D., Jacob, P33, P165 Eugenin, Eliseo, P162 Eyzaguirre, Lindsay, P69 Fields A., Jerel, P25 Fields, Jerel, P42 Filipowicz, Adam, P90 Fischer, Tracy, P46, P71 Fitting, Sylvia, P99 Flores, Ilse, P25, P42 For the Neuropsychology Working Group of the MACS, P112 Fox, Howard, P56 Franklin, Donald, P56 Frieman, Amy, P95 Fry, Kelsi, P61 Fujinami, Robert, P37 Fumaz, R., Carmina, P113 Groma, Valerija, P148 Gruenewald, Analise, P51 Gu, Chao-Jiang, P17, P76 Gu, Chaojiang, P65 Guerrero, Andrea, P167 Gumber, Sanjeev, P52 Gunnam, M., Satya, P46 Guo, Jig, P53 Gupta, Manish, P54 Guy, Joseph, P86 Ha, Seung-Kwon, P86 Hacker, Lindsay, P95 Haddad, Elias, P33 Haile, Woldeab, P158 Hakami, Ramin, P12 Hampson, David, P121 Hanak, Tyler, P37 Hanks, Nancy, P120 Harhaj, Edward, P64 Harrod, B., Steven, P135 Hartman L., Amy, P3 Hartman, Amy, P83 Hashemi, Parastoo, P135 Hassanzadeh, Shiva, P55 Hategan, Alina Popescu, P127 Haughey, Norman, P32, P56, P81 Haughey, J., Norman, P170 Hauser, Kurt, P10, P87, P138, P107 Hauser, F., Kurt, P122, P99 He, Lifan, P15 He, Hongxia, P76 Heaton, Robert, P74 Hellmuth, Joanna, P111 Henderson, Lisa, P57, P58 Herman, Susan, P146 Hershberg, Uri, P125, P109 Hidalgo, Melissa, P106, P105 Hixon M., Alison, P59 Ho, Winson, P39 Hoefer, Melanie, P155 Hongyin, Wang, P17 Hoque, Tozammel, P121 Horvat, Branka, P22 Horvath, Stefan, P131 Hu, Wenhui, P14, P149, P169, P71, P72, P73 Hu, Guoku, P19 Hu, Shuxian, P128, P23 Huang, Xiaofang, P137 Hui, Liang, P60 Hungerford, Laura, P69 Hunter, Meredith, P95 Ibba, Gabriele, P13, P14, P15, P160, P159 Inglese, Matilde, P161 Iordanskiy, Sergey, P12, P61, P62 Iudicello, Jennifer, P63 Jackson, Joe, P166 Jacobson, Steven, P86, P5 Jacobson, Jeffrey, P7, P9, P30, P31, P125, P105 Jacobson, Steven, P47 Jacobson, Lisa, P112 Jaillard, Assia, P119 Jain, Pooja, P33, P47, P48, P64, P137 James, Tony, P7, P31 Jancarik, Andrej, P118 Jaureguiberry-Bravo, Matias, P65 Javandel, Shireen, P111 Jeffrey, Jacobson, P154 Jennifer, Kelschenbach, P17 Jennings, Stephen, P40 Ji, Haiyan, P66 Jiang, Xiong, P55 Johnson, Kory, P39 Johnson, Tory, P58 Johnson, M., Edward, P67 Jones, Dallas, P68 Jones, Simon, P157 Jordan-Sciutto, Kelly, P167, P151 Julius, Fonsah, P74 Jumare, Jibreel, P69 Khalili, Kamel, P13, P14, P54, P71, P72, P73, P15, P169, P140 Khan, Nabab, P79, P80 Khan, Zafar, P64 Khan, K., Zafar, P33, P47, P48, P137 Khuder, Saja, P32, P81 Kiernan, Elizabeth, P75 Kim, Boe-Hyun, P17, P118, P76 Kim, Irene, P51 Kim, SooAh, P81 Kim, Woong-Ki, P87, P90 Kingsley, Lawrence, P171, P112 Kip, Elodie, P82 Knapp, Pamela, P10, P87, P107 Knapp, E., Pamela, P122, P99 Kolson, Dennis, P44, P51, P151 Koneru, Rajeth, P158 Kook, YeonHee, P19 Koralnik, Igor, P21, P115 Koralnik, J., Igor, P146 Krebs, Fred, P7, P11, P31, P125 Kremer, David, P124 Kuate, Callixte, P74 Kucheryavykh, Lilia, P92 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Maki, Pauline, P136, P100 Malcolm, Thomas, P73 Malik, Nasir, P43 Mallard, Jaclyn, P96 Mamik, Manmeet, P8, P97 Mandel, Jordan, P101 Mangus, Lisa, P98 Mankowski, Joseph, P132, P98 Marche, Patrice, P22 Marcondes, G., Maria Cecilia, P77 Marcotte, Thomas, P56, P81 Marks, William, P107 Murovska, Modra, P148 Murray, Jacinta, P17 Murray, Melissa, P171 Nagarkatti, Mitzi, P48, P137 Nagarkatti, Prakash, P137, P48 Nagel, Maria, P68 Naibryf, Tali, P106 Nair, Madhavan, P116 Najera, A., Julia, P77 Nakamura, Christie, P78, P120 Nakasujja, Noeline, P144 Nakigozi, Gertrude, P144 Nambiar, Sreesha, P47 Napoli, Alessandro, P71 Narasipura, Srinivasa, P93, P117 Nath, Avindra, P1, P39, P43, P45, P57, P58, P88, P89, P168, P127 Ndembi, Nicaise, P69 Ndhlovu, Lishomwa, P70, P129 Ndhlovu, C., Lishomwa, P47 Nedelcovych, Michael, P118 Neff, C. Preston, P68 Nehra, P., Artinder, P33 Neumann, Konstantin, P137 Ng, Qimei, P164 Njamnshi, Dora, P74 Njamnshi, Alfred, P74 Noel, Richard, P110 Noggle, Arianna, P96 Nolan, J., David, P96 Nonnemacher, Michael, P154, P4, P7, P9, P11, P24, P30, P31, P125, P109, P103 O'Connor, Erin, P171, P119 O'Donnell, A., Lauren, P84 O’Neill, Alan, P155 Oda, Robert, P78, P120 Ojeda, Daniel, P104 Omeragic, Amila, P121 Otte, Jessica, P72, P143 Oury, Tim, P83 Pendyala, Gurudutt, P123 Peng, Xiao, P75, P91 Perez-Alvarez, Nuria, P114, P113 Periyasamy, Palsamy, P156 Perron, Herve, P22, P124 Peruzzi, Francesca, P85 Phan, Luan, P136 Philip, Ramila, P137 Pillai, Satish, P129 Pirrone Vanessa, P154, P4, P7, P9, P11, P30, P31, P125, P109, P103 Piu, Claudia, P160, P159 Plaud, Marines, P20 Plaud/Valentin, Marines, P126 Pleet, Michelle, P12 Poluektova, Larisa, P153 Poluektova, Y., Larisa, P147 Potash, Mary Jane, P17, P76 Power, Christopher, P8, P133, P97 Prajapati, Bharat, P41 Prasad, Sujata, P23, P128 Prats, Anna, P114, P113 Premeaux, Thomas, P129 Pu, Shelley, P167 Putatunda, Raj, P169 Qin, Xuebin, P75, P91, P169 Qiu, Fang, P74 Qu, Xiying, P66, P169, P130 Quach, Austin, P131 Queen, Suzanne, P132, P98 Rae, Caroline, P157 Ragin, Ann, P112 Raguenez, Gilda, P22 Rais, Rana, P118 Rajan, Neha, P106 Raman, Chander, P48 Rappaport, Jay, P163 Rauw, Gail, P8 Ravina, Kristine, P148 Reich, Daniel, P86 Reiss, Krzysztof, P85 Remirez, Servio, P137 Renard, Felix, P119 Resch, Lothar, P8 Revuri, Nikil, P48 Reynaud, Josephine, P22 Richards, Maureen, P93 Richards, Maureen, P117 Rife, Brittany, P96 Rivera-Morales, Paola, P45 Rivera-Serrano, Mariela, P92 Robertson, Kevin, P144 Rockenstein, Edward, P25 Roda, Weston, P133 Rodrí_guez-Benitez, Rosa, P110 Rodriguez-Benitez, J., Rosa P168 Rodriguez-Valentin, Madeline, P92 Roehm C., Pamela, P15 Roga, Silvija, P148 Romerio, Fabio, P61 Romoli, Benedetto, P77 Rosario, Lester, P134 Roscoe Jr., F., Robert, P135 Roure, Karla Negron, P20 Royal III, Walter, P69 Rubin, Leah, P136, P100 Ruland, Jurgen, P137 Sillman, Brady, P49 Silva, Afonso, P86 Sim, Jordan, P37 Simon, Gary, P138 Singal, Chitra, P41 Singer, Elyse, P131 Singh, Lalita, P44 Singh, Narendra, P48 Singh, Meera, P166 Singh, Vir, P166 Singh, B., Vir, P147 Singh, P., Narendra, P137 Singh, V., Meera, P147 Skolasky, Richard, P126 Skowronska, Marta, P6 Skuja, Sandra, P148 Slusher, Barbara, P118 Song, Jiasheng, P149 Soontornniyomkij, Virawudh, P18 Spitsin, Sergei, P150 Staal, Jens, P82 Steiner, Joseph, P43, P58, P89, P127 Stern, Anna, P151 Stirpe, Nicole, P166 Stoff, David, P152 Strazza, Marianne, P103 Su, Hang, P153 Suin, Vanessa, P82 Sullivan, Neil, P154 Szep, Zsofia, P7, P9, P30, P31, P154, P109 Tagny, Claude, P74 Tanaka, Yuetsu, P5 Tedaldi, Ellen, P71 Tenora, Lukas, P118 Teteris, Ojars, P148 Thaney, Victoria, P155 Thangaraj, Annadurai, P156 Tharakan, Ravi, P56 Thompson, Tiffany , P3 TMARC Group, P139 Tobia, Michael, P157 Toborek, Michal, P6 Traina-Dorge, Vicki, P95 Trapp, Bruce, P124 Tuma, Ronald, P28 Tyagi, Richa, P43, P57, P58, P89, P1 Tyagi, Mudit, P138 Tyler, L., Kenneth, P59 Tyor, William, P158 Ubaida-Mohien, Ceereena, P56 Uleri, Elena, P160, P159 Umlauf, Anya, P69 Valcour, Victor, P111 Valentin, Gabriel, P92 Van Duyne, Rachel, P61 Van Horssen, Jack, P124 VanGucht, Steven, P82 Vatakis, Dimitrios, P51, P131 Veenstra, Mike, P161 Veilleux, Courtney, P162 Velasquez, Stephani, P163 Vellucci, Ashley, P5 Venna, Nagagopal, P115 Victorio, Carla Bianca Luena, P164 Villinger, Francois, P33, P52 Vipulkumar, Patel, P44 Volsky J., David, P17 Volsky, David, P65, P76, P118 Wagner, Wendeline, P163 Walters, Aaron, P83 Wang, Sheng, P32 Wang, Tongguang, P39 Wang, Cuiwei, P55 Wanicek, L., Emma, P165 Ward, Sara Jane, P28 Ward, Jennifer, P158 Weber, Emily, P166 Weber, Kathleen, P136 Wellish, Mary, P95 White K., Martyn, P13, P14, P15 White S., Martyn, P26 White, Martyn, P142, P141 White, G., Alexander, P149 Wigdahl, Brian, P4, P7, P9, P11, P24, P30, P154, P31, P33, P40, P125, P109, P103 Wilk, Anna, P85 Williams, Jean, P4, P109, P125, P154, P30, P31, P7, P9 Williams, Kimberly, P167 Williams, C., Kenneth, P96 Witt, Mallory, P131 Witwer, Kenneth, P133 Wojna, Valerie, P45, P168, P126, P110 Wollebo S., Hassen, P13, P14, P15 Woodson, Caitlin, P61 Woollard, Shawna, P52 Wu, Yuntao, P53 Wurcel, Alysse, P115 Wyczechowska, Dorota, P85 Xu, Yishi, P164 Yagi , Shigeo, P59 Yang, Lu, P19 Yarandi, Shadan, P35 Yen, William, P27, P28 Yen, Cecil, P86 Yen, Po-Jen, P52 Yin, Chaoran, P72, P169 Yoo, Seung-Wan, P32, P170 Young, Mary, P55 Young, Won-Bin, P66, P73, P169, P149, P130 Yu, Guixia, P59 Yu, Fei, P149 Zapata, Adriana, P85 Zeffiro, Thomas, P171, P119 Zhang, Yonggang, P71, P149, P169 Zhang, Ting, P169 Zhao, Huaqing, P169 Zhong, Wen, P7, P9, P30, P31, P154, P125, P109 Zhu, Huanzhang, P66 Zhu, Yu, P97 Zhuang, Zhengping, P39 Zieda, Anete, P148 Zou, ShiPing, P122


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Abstracts from the 14th International Symposium on NeuroVirology October 25–28, 2016, Toronto, Ontario, Canada, Journal of NeuroVirology, 2016, 1-89, DOI: 10.1007/s13365-016-0478-8